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Laboratory for Physiology, Institute for Cardiovascular Research, Vrije Universiteit, Amsterdam 1081 BT, The Netherlands
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hypoxia triggers a mechanism that induces
vasodilation in the whole heart but not necessarily in isolated
coronary arteries. We therefore studied the role of cardiomyocytes
(CM), smooth muscle cells (SMC), and endothelial cells (EC) in coronary
responses to hypoxia (PO2 of 5-10 mmHg).
In an attempt to determine the factor(s) released in response to
hypoxia, we inhibited the contribution of adenosine, ATP-sensitive
K+ channels, prostaglandins, and nitric oxide. Isolated rat
septal artery segments without (
T) and with a layer of cardiac tissue (+T) were mounted in a double wire myograph, and constriction was
induced. Hypoxia induced a decrease in isometric force of 21% and 61%
in T and +T segments, respectively (P < 0.05). EC removal increased the relaxation to hypoxia in T segments to 33% but
had the same effect in +T segments (61%). Only one of the inhibitors,
the adenosine antagonist in +T segments, partially affected the
relaxation due to hypoxia. The role of adenosine is thus limited and
other mechanisms have to contribute. We conclude that hypoxia induces a
relaxation of SMC that is augmented by the presence of CM and blunted
by the endothelium. A single mediator does not induce those effects.
endothelial cells; smooth muscle cells; cardiomyocytes; ATP-sensitive potassium channels; adenosine
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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SEVERAL INVESTIGATORS (1, 2, 4, 6, 10, 20, 21, 27) have shown in isolated whole heart preparations that hypoxia (lowering the PO2 in the perfusion solution) induces vasodilation in the whole heart. However, it is not clear whether or not mediators of hypoxia-induced vasodilation originate from vascular or myocardial tissue.
The results obtained from studies on the effect of hypoxia on isolated coronary artery tone are not consistent and range from dilation or decreases in isometric force (3, 8, 12, 34) to constriction or increases in isometric force (9, 18, 22, 26, 30) or no effect at all (7). Efforts to understand oxygen-sensitive mechanisms by means of studying isolated cells complicated things even more. Several investigators have shown in isolated endothelial cells that hypoxia can increase the production of prostaglandins (17, 33) and nitric oxide (NO) (11, 24) or can decrease the production of prostaglandins (31) and NO (33). Gellai et al. (8) provided evidence for a direct effect of oxygen on coronary smooth muscle cells. These authors showed that contractile function was markedly depressed at a PO2 below 5 mmHg. Myocytes are also sensitive to changes in oxygen tension. Mustafa (19) showed that isolated embryonic cardiac cells from chickens release adenosine and its degradation products in response to hypoxia. Furthermore, Kawaguchi et al. (14) showed that isolated heart tissue from neonatal rats released free fatty acid and prostacyclin during hypoxia.
Thus evidence from the literature suggests that different cell types
that are involved in local flow regulation are sensitive to changes in
oxygen tension. However, based on the studies so far, it is not clear
how each cell type (cardiomyocyte, endothelial cell, and smooth muscle
cell) is involved in the integrated vascular response to hypoxia is
(i.e., when all cell types are present, as in an intact heart).
Therefore, we aimed at developing a new method to investigate the role
of cardiomyocytes, endothelial cells, and smooth muscle cells in
coronary vascular responses to hypoxia. With the use of this method, we
studied the effect of hypoxia on KCl- and U-46619-induced contraction
in coronary artery segments with and without surrounding myocardial
tissue and with and without endothelium. In an attempt to determine the factor(s) released in response to hypoxia, we studied the contribution of adenosine, ATP-sensitive K+ (K
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The animal experimental committee of Vrije Universiteit approved all experiments. Male Wistar rats weighing 250-350 g were anesthetized with 50 mg/kg pentobarbital sodium (Nembutal, Sanofi Sante; Maassluis, The Netherlands). The chest was opened, and the heart was quickly removed and placed in ice-cold (4°C) MOPS-buffered physiological salt solution (PSS) containing (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, 5 D-glucose, 2 pyruvate, 0.02 EDTA, and 3 MOPS; pH was adjusted to 7.35-7.40 with 1 M NaOH. The heart was transferred to a dissecting dish also containing ice-cold MOPS PSS and pinned down to the silicone bottom. The right ventricle was opened, and the right ventricular wall was pinned down to the bottom of the dish, revealing the septum.
Two adjacent segments of the septal artery were dissected with
microscissors. One segment was cleared of connective tissue and
myocytes (T), whereas the other segment was left surrounded by a
layer of 100-200 µm of cardiac tissue (+T). Both segments were
placed in a water-jacketted dual wire myograph setup (made in the
technical department of the Laboratory of Physiology, Vrije Universiteit; Amsterdam, The Netherlands) containing a Krebs
bicarbonate-buffered PSS [containing (in mM) 110 NaCl, 5 KCl, 2.5 CaCl2, 1 MgSO4, 1 KH2PO4, 10 D-glucose, 24 NaHCO3, and 0.02 EDTA] equilibrated with 95% air-5%
CO2; pH 7.35-7.40. The chamber had a volume of 8 ml. Two tungsten wires were inserted through each vessel lumen. One was
connected to a force transducer to measure isometric force, and the
other was fixed to a micrometer to stretch the segment.
Experimental Protocol
The temperature of the tissue bath was increased to 37°C, and vessel segments were equilibrated for a period of 30 min. The rings were then stretched stepwise and at each length exposed to 100 mM KCl Krebs until the induced contraction was maximal (Fig. 1). After the optimal length was determined, segments were set at this length, and the response to 100 mM KCl was repeated to test reproducibility. The segments were then allowed to equilibrate for another 15 min. Endothelial function was tested with 1 and 10 µM acetylcholine. Papaverine (100 µM) was added at the end of the experiment to fully relax the vessel segments.
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After the experiments, the wet and dry weights of the coronary artery segments with surrounding myocardial tissue were measured.
Part 1: preconstriction and endothelium.
In the first part of the study, the contribution of the type of
preconstriction and the endothelium on the effect of hypoxia in T and
+T segments was studied. After equilibration at optimal length, vessels
were precontracted either with an elevated potassium concentration (30 mM KCl, receptor independent) (13) or with 0.001-10
µM of the thromboxane mimetic U-46619 to a comparable force level as
30 mM KCl. U-46619 contracts vessels by activating the inositol
(1,4,5)-trisphosphate/diacetylglycerol pathway
(5). After acetylcholine washout (endothelium
check), vascular responses to 15 min of hypoxia (see
Hypoxia) were tested. To test whether the effect of hypoxia
depended on the endothelium, responses to hypoxia were also studied in
segments without endothelium. Endothelial removal was done by gently
rubbing the intimal surface with rough silicone tubing while the vessel
was still in the tissue to minimize damage to smooth muscle cells. The
procedure was judged successful when the segments failed to reduce
isometric force after application of 1 and 10 µM acetylcholine but
still exhibited a relaxation to 10 and 100 µM adenosine.
Part 2: mediators.
In the second part of the study, we attempted to determine the
factor(s) released in the response to hypoxia. The contributions of
four mediators were investigated in both vessel segments (T and +T).
First, the mediators and inhibitors used are described, followed by the
precise experimental procedure.
Hypoxia
Hypoxia was induced by changing the bubbling of the tissue chamber from 95% air-5% CO2 (PO2
150 mmHg) to 95%
N2-5% CO2 (PO2 5-10 mmHg). Oxygen tensions were measured with a
fast-responding Clark-type oxygen electrode (15), which
was calibrated before and after the experiment. Because this electrode
repeatedly yielded the same PO2 values, we
decided not to measure oxygen tension in all experiments. Some oxygen
could still diffuse into the organ chamber while bubbling with 95%
N2-5% CO2 and, therefore, the oxygen tension
was between 5 and 10 mmHg. After 15 min of hypoxia, the changes in
force generally stabilized and the gas mixture was returned to normoxia.
Viability of the Tissue
To test whether the isolation procedure or the duration of the experiment damaged the vessel segment with surrounding myocardial tissue, two staining methods were used. Three vascular segments were stained before the start of the experiment, and three vascular segments were stained after the experimental protocol.Segments (length of 1.5-2 mm) were cut in half. One half was used to determine peroxidase activity as an index of myoglobin content to check whether the cell membrane of cardiac cells remained intact. When the cell membrane is damaged, myoglobin leaks out of the cell. The other half was used to determine succinate dehydrogenase (SDH) activity as an index of mitochondrial activity.
The peroxidase activity of myoglobin in fixed tissue was determined as described by Lee-de Groot et al. (16). After being fixed, embedded, and frozen, the air-dried sections were incubated, and myglobin staining of the sections was detected qualitatively with a light microscope.
SDH activity was determined in segments that were directly embedded in gelatin, frozen in liquid nitrogen, cut in sections of 10 µm thick, and stained as described by Pool et al. (25). The precipitation of colored formazan, which is formed by the action of SDH on its substrate in the presence of a tetrazolium salt, was measured qualitatively with a light microscope. Staining quality and intensity were assessed blindly by an independent expert in the field of the used methods (16).
Chemicals
Salts, acetylcholine, arachidonic acid, indomethacin, chromakalim, glibenclamide, and 8-PT were obtained from Sigma (St. Louis, MO). Adenosine was obtained from Boehringer-Mannheim. L-NNA was obtained from Bachem (Bubendorf, Switzerland), and DMPX was obtained from ICN. U-46619 was obtained from from Fluka Biochemika. Acetylcholine, adenosine, and L-NNA were dissolved in distilled water. Arachidonic acid was dissolved in ethanol under nitrogen. Indomethacin was dissolved in 0.2 M Na2CO3. Cromakalim, glibenclamide, and DMPX were dissolved in DMSO. 8-PT was dissolved in ethanol and 1 M NaOH (75:25% vol/vol). All the drugs used were added to the superfusion solution and reported as final concentrations.Calculations and Statistical Analysis
Active force (F100) development is defined as the force induced by 100 mM KCl minus the passive force. The force level induced by 30 mM KCl is referred to as the precontraction level (F30). Precontraction level and changes in isometric force induced by agonists or hypoxia are expressed as the percentage of the force induced by 100 mM KCl only to correct for differences in segment length and/or differences in the number of smooth muscle cell layers. Values are reported as means ± SE. We compared the data from segments with and without surrounding tissue or endothelium using a factorial ANOVA. The preconstriction levels of these segments were similar. The effect of inhibitors on precontraction level was assessed by a paired t-test. A general linear model was used to assess the effect of the inhibitor on the response to the agonist or to hypoxia. A Bonferroni correction for multiple comparisons was applied when necessary. A probability value of P < 0.05 was considered significant for all tests.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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A force-length relationship from a typical experiment is shown in
Fig. 1. The vessel characteristics (part 1) are summarized in Table 1. The acetylcholine-induced
decreases in isometric force were significantly larger
(P < 0.05) in T than in +T segments, both for 1 and
10 µM. After endothelial removal, acetylcholine (1 and 10 µM) had
no significant effect on isometric force in both T and +T segments.
In part 2 of the study, 31 vessel pairs were used.
F100 and F30 were not different between T and
+T segments. F30 was 91 ± 2% and 91 ± 3% of
F100 in T and +T segments, respectively. All vessels
responded to acetylcholine, and decreases in force were significantly
larger (P < 0.05) in T than in +T segments, both for
1 and 10 µM. The overall decreases in force due to hypoxia were
17 ± 3% and 56 ± 4% of F100 in T and +T
segments, respectively, and were significantly larger in +T than in T
segments.
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There was no damage of the myocytes close to the coronary artery, and no differences among the three segments that were stained after the experiment compared with segments stained directly after the preparation for both myoglobin and SDH.
Effect of Hypoxia
After equilibration, vessels were precontracted with 30 mM KCl (resulting isometric force is F30) in the buffer solution. Hypoxia induced a decrease in isometric force in bothT and +T segments, which could be repeated at least four times over a time period of 2.5 h, indicating that the preparation was stable. A typical recording (marked with F30) of the effect of
hypoxia on T and +T segments is shown in Fig.
2. Just before N2 (hypoxia) is applied, the preconstriction force level is determined, and just
before O2 (reoxygenation) is applied, the decrease in force due to hypoxia is determined. F100 is the force due to 100 mM KCl at the optimal length, as explained in Fig. 1. In both T and
+T segments, hypoxia induced a reduction in force and significantly more in +T than in T segments; the average results are shown in Fig.
3 (identified as KCl). Hypoxia reduced
isometric force from 90 ± 2% to 69 ± 5% of
F100 in T segments and from 90 ± 2% to 29 ± 5% of F100 in +T segments (P < 0.05 vs.
T segments, n = 11). There was no relation between
the wet or dry weights of +T segments and the effect of hypoxia.
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Effect of Hypoxia and Type of Preconstrictor
In Fig. 3, the effect of hypoxia in U-46619 (0.003-1 µM)-preconstricted vessels is also shown. The force levels normalized to F100 are 88 ± 3% and 83 ± 5% forT and
+T segments, respectively, and are similar to the 30 mM KCl-induced
force levels. Hypoxia reduced force significantly to 72 ± 5% and
to 34 ± 5% for T and +T segments, respectively
(n = 11, P < 0.05). The reduction in isometric force induced by hypoxia during U-46619-induced contraction was not significantly different from the reduction in KCl-induced contraction in both T and +T segments but significantly larger in +T
than in T segments (P < 0.05) for both constrictors.
Effect of Hypoxia After Endothelial Removal
After endothelial removal (Fig. 4), hypoxia reduced force inT segments from 89 ± 2% to 56 ± 8% (n = 6, P < 0.05). In +T segments,
hypoxia reduced isometric force from 90 ± 6% to 29 ± 8%
(n = 6, P < 0.05), significantly more
than in T segments (P < 0.05).
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ANOVA revealed a significant effect of both tissue and endothelium. The
relaxation to hypoxia is significantly larger in T segments without
endothelium than in T segments with endothelium (post hoc
t-test, P < 0.05). However, endothelium
removal had no effect on the response to hypoxia in +T segments (post
hoc t-test, P > 0.05).
Mediators in the Response to Hypoxia
For the second part of the study, 31 pairs of vessels were used. In all cases, F100 was determined and used to correct (force levels as a percentage of F100) for differences in segment length and/or the number of smooth muscle cell layers. In group 1, the effect of the nonselective adenosine receptor antagonist 8-PT on the response to hypoxia was tested (Fig. 5). In the absence of 8-PT, 30 mM KCl induced a precontraction level of 78 ± 6% and 95 ± 4% inT and +T segments, respectively. After the addition of 8-PT, these
force levels were 73 ± 6% (P = 0.27 vs. control) and 65 ± 5% (P < 0.01). The response to
exogenous adenosine on the precontracted segments in the absence and
presence of 8-PT is shown in Fig. 5B. In the presence of
8-PT, the relaxation due to adenosine was significantly reduced,
implying that the inhibitor is effective in our preparation. Finally,
the response to hypoxia is shown in Fig. 5C. The response to
hypoxia was larger in both the absence and presence of 8-PT in +T than
in T segments (P < 0.01 and P = 0.02 for control and 8-PT, respectively). The response to hypoxia in +T
segments was significantly reduced by 8-PT.
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The results of the other inhibitors (groups 2-5) are
summarized in Table 2.
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Table 2 is organized similarly to Fig. 5. The preconstriction force due
to 30 mM KCl, the relaxation of the mediator, and finally the
relaxation due to hypoxia was always without (control) and with
inhibitor. An effect of DMPX on preconstriction force was found
in T and +T segments and an effect of L-NNA was found in
T segments. All relaxations due to the used mediators were blocked with the appropriate inhibitor, indicating that each of the
inhibitors is functional in our setup. None of the inhibitors affected
the relaxation due to hypoxia.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study shows that two segments of the isolated rat coronary artery, one with and one without a layer of myocardial tissue, mounted in a double wire myograph is a suitable setup for studying the effect of hypoxia on myocardial tissue and the embedded coronary artery.
The segments relaxed differently to the endothelium-dependent dilator acetylcholine. Both segments relaxed in response to a change in oxygen tension from 150 to 5-10 mmHg, but relaxation in segments with cardiac tissue was stronger under all conditions studied. In coronary arteries without and with surrounding myocardial tissue, the effect of hypoxia is the same in 30 mM KCl- or U-46619-constricted segments. Endothelial removal increased the relaxation to hypoxia only in artery segments without surrounding myocardial tissue.
Experiments with inhibitors of potential mediators of the hypoxic
response show that K
Preparation
To investigate the contribution of the endothelium, smooth muscle cells, and cardiac myocytes to hypoxia-induced vascular changes and the role of mediators in this response, we developed a new preparation. By simultaneously studying two isolated segments, one without and one with a surrounding layer of cardiac tissue, and by comparing the effect of hypoxia on the tone of both segments, the contribution of cardiac myocytes to the response can be quantified. Then, by removing the endothelium, the contribution of the endothelium to the hypoxic response can be quantified. Although the absolute level of 100 mM KCl-induced constriction was reduced in endothelium-denuded vessels (see Table 1), the 30 mM KCl-induced contraction, expressed as a percentage of F100, was not changed, because the absolute level of the 30 mM KCl-induced contraction was also reduced. The acetylcholine response in Table 1 is smaller in the vessel with surrounding tissue and may be due to enzymes present in the myocardial tissue that effectively lower the acetylcholine concentration.We are the first to use an isolated vessel segment surrounded by a thin layer of cardiac myocytes, and therefore we performed some histochemical analysis of SDH concentration (25) and myoglobin concentration (16) of the preparation to establish the mitochondrial viability and membrane integrity of the tissue. The SDH and myoglobin staining intensity of the myocytes surrounding the vessel segment showed no signs of damage both directly after preparation and after termination of the experiment (except for the myocytes at the cutting edge of the preparation). Thus the cardiac myocytes were viable throughout the experiment.
The absolute amount of myocardial tissue is no determinant of the response to hypoxia. We measured both the wet and dry weights of the coronary artery segments with surrounding myocardial tissue and correlated these values with the effects of hypoxia on isometric force. There was no correlation, suggesting that the amount of myocardial tissue did not affect the response to hypoxia.
Furthermore, we calculated the amount of oxygen consumption of the layer of myocardial tissue, assuming a solid cylinder (worst-case scenario) of myocardial tissue under resting conditions based on oxygen diffusion coefficients. The maximal decrease of oxygen tension was 3 mmHg in the center of the cylinder, indicating that the development of an anoxic core near the center of the preparation is unlikely.
Role of the Endothelium
Our results clearly show that the relaxation in response to hypoxia is not caused by a dilatory factor released by the endothelium, because endothelium removal did not abolish the decrease in force induced by hypoxia. However, the relaxation induced by hypoxia is larger in endothelium-denuded vessels. This might be caused by a hypoxia-induced constrictor mechanism present in the endothelium. Indeed, several studies (30, 34) on coronary preparations have shown that a hypoxic contraction was abolished by endothelial removal. In +T segments, removal of the endothelium had no significant effect on the response to hypoxia. This could be caused by the effect of a mediator released from the endothelium that has an effect on the contractile state of the surrounding cardiac myocytes (28) that counteracts the effect of endothelin on the contractile state of the vessel. In addition, the myocyte itself responds to hypoxia and releases substances that regulate the secretion of endothelin and a relaxant by the endothelial cells (32). Thus a complex cross-talk between myocytes and endothelium exists.Smooth Muscle Cells
The observation that endothelial removal in the segment without surrounding tissue does not inhibit the response to hypoxia suggests that changes in oxygen tension have a direct effect on the smooth muscle cells. According to Paul (23), the total phosphagen pool in smooth muscle cells is small compared with its utilization; energy supply for contraction relies for the greater part on continuous intermediary metabolism. In this process, oxygen is required, and a reduction in oxygen availability will impair intermediary metabolism and contraction.The effect of hypoxia on vascular relaxation appears independent of the type of constrictor used. Smooth muscle cells were activated by depolarization with a 30 mM KCl PSS, increasing the open probability of the voltage-sensitive Ca2+ channels (13). U-46619, a thromboxane mimetic, was used to stimulate smooth muscle cells in a receptor-dependent manner, activating the inositol (1,4,5)-trisphosphate/diacetylglycerol pathway (5). Thus the effect of hypoxia is independent on the intracellular pathway of contraction, and without both tissue and endothelium, there is a significant relaxation, indicating a role for the smooth muscle as a sensing element for oxygen.
Myocardial Tissue
The decrease in isometric force due to hypoxia (Figs. 2 and 3) is more in segments with a layer of cardiac myocytes. This strongly supports that the myocardial tissue releases a factor in response to hypoxia that reduces isometric force. Figure 3 shows that the additional relaxation (difference in relaxation between +T andT
segments) due to the surrounding myocardial tissue is larger than the
relaxation of the vessel alone (T segments). It should be noted that
the energy need of the cardiac tissue in our preparation is primarily
determined by basal metabolism, whereas in actively contracting tissue,
the energy need is also determined by excitation-contraction coupling
metabolism and mechanical activity associated with cross-bridge
turnover (29). Therefore, the contribution of the cardiac
myocytes in the beating heart is expected to be stronger. It seems that
in +T with low metabolic activity, the large relaxation in response to
hypoxia leaves little capacity to relax even more should the myocytes
contract. However, it should be noted that the change in the oxygen
tension used in this study is quite dramatic (from 150 to 5-10
mmHg). In the working heart, changes in oxygen tension induced by
changes in activity are smaller.
Inhibitors
Prostaglandins and NO are not the mediators of the relaxation in both arteries without tissue and with tissue. Furthermore, the mechanism of relaxation is not through opening of KIn conclusion, we developed a method to study the interaction between myocardial tissue and the vessel wall. We have also shown that the response to hypoxia is independent of the type of constrictor used. The vasoactive effects of hypoxia are not mediated by a single uniform mechanism but are probably interplay of several mechanisms that are activated under different conditions. The surrounding myocardial tissue is the dominant regulator of vascular tonus changes under conditions where the oxygen supply is impaired.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Willem van der Laarse for expert help in assessing the staining quality and intensity of the preparations for myoglobin and succinate dehydrogenase. The authors are grateful to Dr. Nico Westerhof for comments and valuable suggestions regarding the manuscript.
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FOOTNOTES |
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First published November 15, 2001;10.1152/ajpheart.00179.2001
This study was supported by Netherlands Heart Foundation Grant 93-103.
Address for reprint requests and other correspondence: P. Sipkema, Laboratory for Physiology, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands (E-mail: sipkema{at}physiol.med.vu.nl).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 March 2001; accepted in final form 9 November 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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significantly larger reduction in isometric force than
that in 