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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 5, 2001 as doi:10.1096/fj.00-0497fje. |
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Institut für Herz-und Kreislaufphysiologie, Heinrich-Heine-Universität Düsseldorf, Germany
2Correspondence: Institut für Herz-und Kreislaufphysiologie, Heinrich-Heine-Universität Düsseldorf, Postfach 101007, 40001 Düsseldorf, Germany. E-mail: schrader{at}uni-duesseldorf.de
SPECIFIC AIMS
The recent generation of myoglobin (Mb) knockout (myo-/-) mice with their surprisingly benign phenotype presents a new challenge to Mb’s functional relevance. Although previous studies in transgenic mice demonstrated that chronic lack of Mb triggers compensatory mechanisms, the present study was aimed at exploring the acute blockade of Mb by CO in isolated hearts with myo-/- mice serving as appropriate controls. This approach enabled us to 1) address the question of CO’s specificity as an inhibitor of Mb; 2) quantify the role of Mb as oxygen store in the beating mammalian heart; 3) test the hypothesis of Mb-mediated oxidative phosphorylation; and 4) demonstrate Mb’s functional relevance in intracellular O2 supply by facilitating O2 diffusion.
PRINCIPAL FINDINGS
1. Mb’s O2 reservoir is of functional relevance during
short periods of ischemia
To assess the functional relevance of the
MbO2 buffer in the mammalian heart, Langendorff
perfused hearts from myo-/- and wild-type (WT)
mice were subjected to brief no-flow ischemia. Cardiac function
decreased more steeply in myo-/- hearts,
especially during the first seconds of ischemia. After 2 s of
ischemia, dP/dtmax had fallen by 9.4 ±
2.8% in the knockout versus 5.4 ± 2.1% in the WT
(P<0.05). Thereafter, a more parallel pattern of functional
decline developed. Significant differences of similar magnitude were
also seen for left ventricular developed pressure (LVDP) and
dP/dtmin (P<0.05). After acute CO
inhibition of Mb, the ischemia-induced decrease in cardiac function in
WT hearts was identical to the effect of chronic Mb lack in hearts from
myo-/- mice. Myo-/- hearts
were unresponsive to the application of CO.
2. Acute inhibition of Mb with CO impairs contractile force
development when Mb is partially deoxygenated
To approximate conditions that might mimic the situation in
vivo with respect to Mb, we progressively lowered the
PO2 in the perfusion medium by equilibrating it
with O2 ranging from 95% to 12%. At the same
time, the extent of Mb oxygenation was measured by
1H NMR spectroscopy. This approach enabled us to
determine the intracellular MbO2 dissociation
curve within the beating heart. Mb desaturation first appeared at 65%
arterial O2 when Mb was desaturated by 13 ±
3%, the lowest measured MbO2 saturation of
18.6 ± 5% was reached at 12% arterial O2.
With the range at which Mb desaturation occurs in the perfused beating
heart defined, the effect of CO on cardiac performance and
O2 utilization in myo-/-
and WT mice was studied at different levels of
MbO2 saturation, ranging from 100% to 51%. When
Mb was fully O2-saturated (arterial medium
equilibrated with 75% O2), WT and
myo-/- hearts showed no significant change in
functional parameters as well as coronary venous
PO2
(PvO2) and thus
O2 consumption (VO2). Under
conditions resulting in partially saturated Mb, however, WT hearts
responded to CO with decreased VO2 and a decrease
in LVDP, whereas myo-/- hearts were always able
to sustain LVDP with unaltered or even slightly increasing
VO2. This effect was most pronounced at 87%
MbO2 saturation (arterial
O2 at 65%). As shown in Figure 1
, myo-/- and WT hearts exhibited opposite
responses when subjected to 20% CO (arterial O2
at 65%). WT hearts revealed a highly significant increase in
PVO2
(PVO2, 28.8±6.4 mmHg 
36.7±5.6 mmHg, P <
0.001) accompanied by a decrease in LVDP of 11% (P < 0.001). In
contrast, myo-/- hearts displayed a slight decrease in
PvO2 (36.8±4.5 mmHg
34.7±4.6 mmHg, P<0.05) and were able to maintain LVDP.
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3. O2 consumption is decreased in cardiomyocytes from
Mb knockout mice
To investigate whether the observed differences in the rate of
VO2 can also be demonstrated at the cellular
level, isolated cardiomyocytes (CM) from WT and
myo-/- hearts were analyzed at defined
PO2. VO2 of freshly
isolated, stimulated mouse CMs was found to be 14.5 ± 1.5 nmol
O2·min–1·mg
protein–1 (n=4), and identical values were
determined in myo-/- CMs (14.6±1.0 nmol
O2·min–1·mg
protein–1; n=3). As illustrated in Figure 2
, reduction in ambient PO2 from 40 mmHg to 8mm Hg
did not change VO2 of WT myocytes, whereas
PO2 values below 8 mm Hg led to a rapid decline
of VO2. In myo-/- CMs, the
onset of VO2 decrease was observed at a higher
PO2 and VO2 reduction was
more pronounced. Thus VO2 was significantly
decreased at low ambient PO2 in
myo-/- compared with WT CMs (i.e., at 10mmHg
ambient PO2: 57±15 vs. 89±10% of baseline,
respectively). The width of myo-/- CMs was
smaller than that of WT CMs (4.8±2.1 µm vs. 5.4±2.6 µm, n=133 and
163, respectively, p<0.001), whereas length did not differ (74±21
µm vs. 71±18 µm, n=133 and 163, respectively, n.s.).
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CONCLUSIONS
We found that acute inhibition of Mb under conditions of minor Mb
deoxygenation leads to significant impairment of left ventricular
contractility and reduced myocardial O2
consumption. In conjunction with our studies on the
O2 uptake of stimulated cardiomyocytes, these
findings provide conclusive evidence that Mb facilitates the diffusion
of O2 from the vasculature to mitochondrial
cytochromes in the beating heart (Fig. 3
). We demonstrate that for Mb to exert this function, it must be
desaturated by only about 10%, a value within the range found in
blood-perfused hearts. We also quantified the role of Mb as
O2 reservoir and tested the hypothesis of
Mb-mediated oxidative phosphorylation. Although Mb appears important
during brief periods of ischemia, our data do not support a direct Mb
effect on oxidative phosphorylation in the mouse heart.
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Role of myoglobin as O2 store
Lack of Mb significantly steepens the ischemia-induced decline in
left ventricular force development. This effect was most pronounced
within the first 2 s of ischemia. Because myocardial Mb content
(0.19 mmol of Mb/kg wet weight), its O2 storage
capacity (1 mol O2/mol Mb) and
VO2 are known, the maximum period during which
O2 released from Mb can support myocardial
oxidative metabolism is calculated at 2.8 s. This value is
consistent with our experimental findings. In addition to tiding the
heart over from contraction to contraction, the advantage of such a
delay in functional decline may be relevant during short periods of
tachycardic arrhythmia resulting in a relative decrease in diastolic
coronary perfusion.
Myoglobin-mediated oxidative phosphorylation has been proposed as a mechanism to support ATP generation by cardiac cells under conditions of fully oxygenated Mb. As an underlying mechanism, a preferred uptake of Mb-bound O2 by mitochondria and/or the acceptance of electrons by sarcoplasmic Mb with concomitant reduction of heme iron ligated O2 to H2O have been suggested. According to this hypothesis, one would expect myocardial VO2 to be decreased in hearts lacking Mb or following acute blockade of Mb. However, we found no significant functional differences between isolated myo-/- and WT hearts. Furthermore, acute inhibition of Mb by CO at 75% arterial O2 had no effect on myocardial VO2 and contractile parameters; although the perfusion-medium was of sufficiently high PO2 to fully oxygenate sarcoplasmic Mb. Thus, we discovered no evidence for Mb-mediated oxidative phosphorylation in the mouse heart.
Myoglobin-facilitated O2 diffusion
This study establishes that acute inhibition of Mb with CO under
conditions of partially deoxygenated Mb results in a significant
decrease in VO2 and contractility (Fig. 1)
. This
effect is specific for Mb, because no functional changes were observed
in myo-/- hearts under otherwise identical
conditions. In addition, contracting CMs from
myo-/- hearts were characterized by lower
VO2 at any given ambient
PO2 below 15mm Hg (Fig. 2)
, which clearly
indicates an increase in the apparent diffusive resistance to
O2. This increase in resistance was observed
although cell width in myo-/- CMs was
significantly reduced (4.7 vs. 5.2 µm), which decreased diffusion
distances for O2. Together these findings
demonstrate that Mb is essential for the delivery of
O2 from the sarcolemma to the mitochondria by way
of Mb-facilitated O2 diffusion.
To assess the relative contribution of Mb-facilitated O2 diffusion versus the diffusion of physically dissolved oxygen to intracellular O2 flux, several factors affecting these two parallel pathways have to be considered. Mass flux via diffusion in general is governed by a) the concentration difference (‘gradient’), b) the diffusion coefficient, and c) the diffusion distance. It follows that the quantitative extent of Mb-mediated O2 diffusion depends primarily on the intracellular concentrations of O2 and MbO2 as well as on the in vivo diffusion coefficients of O2 and MbO2, respectively.
The average intracellular concentrations of O2 and MbO2 differ by at least one order of magnitude. With the assumption of an intracellular PO2 of 10 mm Hg, the concentration of physically dissolved O2 is about 13 µM. This finding compares to a myoglobin concentration of 190 µM determined in the mouse heart in the present study. Under well-oxygenated conditions, most of the myoglobin will be oxygenated (see below).
The difference in concentration of O2 and MbO2 favoring Mb-facilitated diffusion is counterbalanced by the diffusion coefficient of physically dissolved O2 (DO2), which is up to 100-fold higher than the diffusion coefficient of Mb (DMb) (aprox. 2x10–5 cm2/s for DO2 vs. 2–23x10–7 cm2/s for DMb). Unfortunately, precise in vivo data for both DO2 and DMb are difficult to obtain with conflicting data being reported for both values. An increase in the ratio DMb/DO2 would result in a higher fraction of O2 supply by Mb-facilitated O2 diffusion and vice versa.
Intracellular O2 diffusion is governed not by the average concentration of O2 or MbO2, but by the respective concentration differences between the site of O2 supply (the sarcolemma) and the site of O2 consumption (the mitochondria). Therefore, the concentration gradients for O2 and MbO2 have to be considered. Under conditions of a fully oxygenated Mb, that is, in the absence of a MbO2 concentration gradient at a high intracellular PO2, theory predicts that Mb-facilitated O2 diffusion does not contribute to the intracellular O2 flux to any significant extent. In line with this prediction, inactivating Mb with CO at high arterial PO2 was without functional effects on the saline-perfused heart. Under these conditions, O2 flux apparently was solely dependent on the diffusion of physically dissolved O2. In contrast, when arterial O2 supply was lowered and intracellular Mb partially deoxygenated, blocking Mb by CO significantly reduced VO2, which demonstrates the importance of Mb-facilitated O2 diffusion in presence of a MbO2 gradient within the CM.
When comparing WT and myo-/- hearts, note
that the diffusion distance is not identical. In a previous study, we
showed that the capillary distance was reduced in
myo-/- hearts. The present study demonstrating a
reduced width of CMs complements this finding. It emphasizes that in
the case of Mb deficiency nature has optimized diffusion distances to
boost O2 delivery by simple diffusion. Along with
this interpretation, the highest Mb content has been reported in muscle
fibers that exhibit the greatest diffusion distances. In this context,
it is remarkable that O2 consumption was lower in
myo-/- CMs despite shorter diffusion distance
(Fig. 2)
, which demonstrates that the reduction in cell width is not
sufficient to compensate for the loss of Mb-facilitated
O2 diffusion.
Functional implications
Mb-facilitated diffusion clearly played a substantial role
in intracellular O2 transport when ambient
PO2 was low (8 mm Hg as seen in isolated
cardiomyocytes) and myoglobin partially deoxygenated (13% Mb vs. 87%
MbO2, as shown in the saline-perfused heart).
Whether similar conditions exist in vivo has been a matter
of debate for decades. 1H NMR spectroscopy
studies in striated muscle of exercising humans unequivocally
demonstrated that MbO2 desaturates with exercise;
that is, that cellular PO2 is low enough for
unloading of O2 from MbO2.
Thus, parallel diffusion of physically dissolved
O2 and Mb-bound O2 is
likely to occur. Several groups have also addressed the question of
cardiac MbO2 saturation on the whole organ level
in vivo. Based on data obtained by scanning reflectance
spectroscopy, average MbO2 saturation was
calculated to be in the order of 92%. It is thus most likely that
deoxygenated Mb (and thus a MbO2 concentration
gradient) do exist in the blood-perfused heart. Because the present
study demonstrated Mb-mediated facilitated diffusion of
O2 in the presence of minor Mb deoxygenation,
this effect appears to be functionally relevant also under in
vivo conditions. When considering the results reported here, we
would expect the delivery of oxygen via Mb-facilitated diffusion to
become even more important under conditions of reduced
O2 supply (e.g., coronary artery disease) or
extended diffusion distance (e.g., cardiac
hypertrophy).
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0497fje ; to cite this
article, use (February 5, 2001) FASEB J. 10.1096/fj.00-0497fje 
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