The purpose of the present study was to directly visualize radial gradients of intracellular in a single individual cardiomyocyte isolated from the rat ventricle. Microspectrophotometry with the use of cytosolic myoglobin as an oxygen probe was conducted at 410 nm. When the quiescent cell was incubated with 1 μM carbonyl cyanidem-chlorophenylhydrazone to increase oxygen consumption approximately eightfold, gradual decreases in myoglobin oxygen saturation (SMb) were demonstrated toward the core of the cell, whereas these decreases disappeared when the cell was treated with 2 mM NaCN. These results highlighted the importance of diffusional oxygen transport in determining intracellular oxygenation in cardiac cells. From the measured SMb, we assessed the profile of radial changes in intracellular at the mean SMb comparable to that in vivo (∼0.5). Quite steep gradients were demonstrated in the vicinity of the sarcolemma that were rapidly attenuated toward the cell core. These radial profiles of intracellular demonstrate the significance of myoglobin-facilitated diffusion of oxygen. Furthermore, the shallow gradients of near the center of the cell might arise from partial depression of oxygen consumption near the cell core.
- oxidative metabolism
- hypoxic core
recent spectrophotometric measurements of fractional oxygen saturation of myoglobin (SMb) in near maximally exercising red skeletal muscles have indicated significant gradients from capillary blood to the sarcolemma (6, 7, 10, 26). These gradients appear so steep that drops at a rate of ∼7 Torr/μm even at moderate oxygen consumption (V˙o 2) as the oxygen molecule travels a distance <2 μm from the erythrocyte to the cell surface (10). In contrast, cryospectrophotometric determination of SMb of the blood-perfused working heart in vivo (5) and 1H NMR study of the isolated, perfused rat heart (14) reported much larger extracellular gradients ranging from 15 to 30 Torr/μm even at normal restingV˙o 2.
Because of the presence of surprisingly large extracellular gradients, at the sarcolemma may decrease to that at approximately half-saturation of myoglobin (P50; 5.3 Torr at 37°C) in the beating heart (5). Hence, the intracellular gradient from the sarcolemma to the mitochondrial membrane is a factor that critically determines oxygen transport to mitochondria and, therefore, oxidative phosphorylation. The magnitude of intracellular gradients of the cardiomyocyte has been estimated from the simultaneous measurements of mitochondrial and cytosolic by using two separate intracellular oxygen probes (i.e., mitochondrial enzymes and myoglobin) in a suspension of isolated cardiomyocytes. Previous studies demonstrated quite shallow gradients between these compartments [<2 Torr at maximalV˙o 2 (13, 24)], probably due to facilitation of oxygen diffusion by an intracellular oxygen carrier, myoglobin (25-27). If so, intracellular of the normal beating heart would be substantially lower than that of the capillary blood but still high enough to sustain mitochondrial oxidative phosphorylation. These results seem to assign a minor role to the diffusional oxygen transport in the regulation of oxidative phosphorylation in the heart, at least at a moderate work rate.
Other than the gradient between the cytosol and the mitochondria, gradual changes perpendicular to the capillary direction may be present in the intracellular space (radial gradients). The magnitude and physiological significance of the radial intracellular gradients have not been fully determined in the cardiac cell. We postulated that the radial gradients might be a factor that limits oxidative phosphorylation in the cardiac cell at increased oxygen demand or during hypoxia (20). We undertook the present study to directly quantitate the intracellular radial gradients in a single quiescent cardiomyocyte isolated from the rat when cellular oxygen demand was significantly increased.
Prior approval for the experiment was obtained from the Animal Research Committee, Yamagata University School of Medicine.
Single cardiomyocytes were isolated from the ventricles of the pentobarbital sodium (50 mg/kg ip)-anesthetized adult Sprague-Dawley rat using the collagenase (type II, Worthington, Freehold, NJ) digestion method, as described previously (21). Isolated cardiomyocytes were suspended in a HEPES buffer solution containing (in mM) 150.0 NaCl, 3.8 KCl, 1.0 KH2PO4, 1.2 MgSO4, 10.0 glucose, and 10.0 HEPES (pH adjusted to 7.35 at room temperature) supplemented with 0.1% BSA. Extracellular Ca2+concentration was 1.0 mM except for the experiment using carbonyl cyanide m-chlorophenylhydrazone (CCCP), in which the buffer solution was Ca2+ free.
High spatial resolution spectrophotometry was conducted in a single individual cardiomyocyte using cytosolic myoglobin as an intrinsic oxygen probe. The previously described measuring system and image processing technique (20, 21) have been modified. The present measuring system consists of a light source, a computer-controlled monochrometer, and a microscope equipped with a digital charge-coupled device (CCD) camera. Light emitted from a direct current-powered 60-W metal halide lamp (LA-60Me, Hayashi Clock Works, Tokyo, Japan) was introduced to a computer-controlled monochrometer (SPG-100ST, Shimadzu, Kyoto, Japan). Monochromatic light at 410 nm (bandwidth 3 nm) was directed to a microscope (BH-2, Olympus, Tokyo, Japan), and the transmitted image of a single individual cell, via a ×40 objective lens (numerical aperture = 0.55; LWDCDPlan ×40, Olympus), was captured by a 10-bit digital CCD camera (C4742, Hamamatsu Photonics, Hamamatsu, Japan). Captured cell images were stored in a computer and subsequently processed as described below. We carefully selected the wavelength for the spectrophotometry to fulfill the following requirements:1) the wavelength corresponds to the Soret band of oxymyoglobin, and 2) the spectrum of the light source is relatively flat around the selected wavelength. The spatial resolution of the final output image on the computer monitor was 0.14 μm/pixel. Image analysis software (IPLab, Signal Analytics, Vienna, VA) controlled the monochrometer and CCD camera.
A 15-μl cell suspension containing ∼1,500 cells was placed on the poly-l-lysine (Sigma, St. Louis, MO)-coated glass slide of an airtight measuring cuvette that provided gas inlet and outlet ports. The measuring cuvette was then transferred to the stage of the microscope and was connected to the outlet port of a computer-controlled gas blender consisting of mass-flow controllers (SEC-320, STEC, Kyoto, Japan). The gas blender supplied a mixed gas of desired oxygen concentration via a humidifier at 2 ml/min. We continuously monitored at the gas outlet port of the measuring cuvette using a conventional oxygen electrode (model 17026, Instrumentation Laboratory, Lexington, MA).
First, the cell suspension was superfused with 99.999% N2 gas, and the transmitted cell image was captured and stored in the computer. The digitized cell image was designated asY deoxy. The fractional concentration of oxygen in the superfusing gas was then raised to either 2.09, 3.14, or 4.09%, and the cell image (Y) was captured again. Finally, the cell was completely oxygenated by superfusion with 21% O2 gas, and the cell image (Y oxy) was captured for a third time. Unless otherwise noted, the cell was incubated with 1 μM CCCP to significantly augment the intracellular gradients (seeV˙ o 2 measurement). All measurements were conducted at 27°C. Because cell images at three different were needed for reconstruction of intracellular oxygenation, one complete measurement required a period of ∼7 min. We selected relatively large myocytes in which intracellular gradients may be exaggerated.
Spectrophotometric determination of SMb.
We conducted digital image processing on the captured cell images to determine the light absorption of the cell with a subcellular spatial resolution. The basic assumption was that changes in light absorption associated with changes in oxygenation level of the cell are exclusively attributable to changes in light absorption of cytosolic myoglobin and thus report cytosolic oxygenation. Before the cell image was digitized, the analog circuit in the video amplifier (C4742) subtracted a part of the light intensity signal (voltage corresponding to the transmitted light intensity that is not affected by changes in myoglobin light absorption) and amplified the remaining part of the signal. This procedure is mathematically represented as Equation 1 whereXij andYij denote the values for a pixel [coordinate (i,j)] of the transmitted cell image and the final digitized cell image, respectively, and α and β are gain and offset of the video amplifier, respectively. According to the Beer-Lambert law, light absorption by intracellular pigments can be represented as Equation 2where Iij is the intensity of incident light; ε and Cij denote the molar absorption coefficient and the myoglobin concentration at pixel (i,j), respectively; superscripts oxy and deoxy refer to oxygenated and deoxygenated myoglobin, respectively;Mij represents light absorption by intracellular pigments other than myoglobin; andLij is the length of the light path. By taking advantage of the fact that light absorption in a single cardiomyocyte is extremely small,Eq. 2 can be linearized as Equation 3wherek is a constant. Finally, the following calculation conducted for each pixel converts the transmitted light intensity to SMb at coordinate (i,j) Equation 4
The position of each pixel relative to the cell must be identical for all three separately obtained images. To avoid error arising from slight movement of the cell during image acquisitions, we aligned the three cell images, if necessary, before calculation of SMb as follows. First, we chose five to seven marker points in a small region of interest (ROI) that were peculiar with respect to light absorption, and the absolute positions of these points were recorded. These marker points appeared to correspond to locations of mitochondria according to the results of rhodamine-123 staining (data not shown). We then checked whether these marker points were in fact found at the same absolute coordinates in the remaining two images. If not, the deflections of the markers from the expected coordinates were calculated. Finally, the affine transform (linear mapping of an image including shifts and rotation) was conducted over the remaining two cell images so that the square errors of the deflections could be minimized. Also, the images were low-pass filtered three times.
After the alignment and low-pass filtering, the calculation depicted inEq. 4 was conducted for all the pixels. We determined the local SMb as follows. First, we arbitrarily selected, in the SMbimage, ∼12 × 4-μm rectangular ROIs within a cell parallel to the long axis of the cell (see Fig. 3). An SMb distribution histogram was then calculated for each ROI. The histogram was subsequently fitted to a normal distribution using IgorPro data analysis software (WaveMetrics, Lake Oswego, OR). In cases in which the histogram was significantly skewed or the standard deviation of the SMb histogram was >0.5, we discarded the data. Finally, we calculated the mean of the histogram and regarded it to represent the local SMb.
We conducted a calibration that relates local SMb to local . We added 2 mM NaCN to the suspension medium and conducted SMb measurements for superfusion gas containing either 0.25, 0.51, 0.96, 2.09, or 3.14% oxygen. We assumed that NaCN abolishes the consumption of oxygen by the cell, thereby abolishing gradients from the extracellular medium to the intracellular space. Hence, intracellular is in equilibrium with gas . The relationship between of the superfusion gas and measured SMb was fitted to the Hill equation.
Because the magnitude of intracellular gradients would be proportional to flux of oxygen to the cell (10), we used 1 μM CCCP (an uncoupler of oxidative phosphorylation) to amplify the intracellular gradients. Therefore, we needed to determine V˙o 2 of the cell in the presence of 1 μM CCCP. Five milliliters of the cell suspension were placed in the airtight measuring cuvette, in which an oxygen electrode (model 17026, Instrumentation Laboratory) was inserted. The cell suspension was vigorously stirred using a magnetic stirrer. The time-dependent decrease in of the suspension medium was recorded, and the rate of fall of ( /Δtin Torr/min) was converted to the rate of oxygen consumption (nmol O2 ⋅ min−1 ⋅ 106cells−1) using the following equation whereN is the number of cells per milliliter and βw is the solubility of oxygen in water (1.62 μmol ⋅ l−1 ⋅ Torr−1at 27°C).
We attempted a visualization of intracellular oxygen with a subcellular spatial resolution in the presence of an uncoupler of oxidative phosphorylation, 1 μM CCCP. When the cell suspension was superfused with 2.09 (15.2 Torr) or 3.14% (22.8 Torr) O2 gas, intracellular SMb averaged over the cell was ∼0.4–0.7 (corresponding to ∼1.9–8.7 Torr; see the results of calibration below) (Fig. 1, solid curve). These results indicate the presence of large gradients in the extracellular medium, presumably resulting from the absence of the specific oxygen carrier myoglobin and the unstirred layer surrounding the cell surface (18). It should be noted that intracellular oxygenation in this condition appears comparable to the volume-averaged SMb reported in the working cardiac cell in vivo (3, 5).
Figure 2 shows the representative data demonstrating the visualization of intracellular oxygenation in a single individual cardiomyocyte. For of the superfusion gas of 15.2 Torr, significant gradients of SMbfrom the sarcolemma toward the center of the cell were demonstrated (indicated in pseudo colors). To quantitatively analyze these radial heterogeneities of SMb, we calculated SMb in small rectangular ROIs within a cell (Fig.3 A). Histograms of the SMb value in these ROIs were generated (Fig.4 A) and fitted to normal distributions, and the mean SMb was determined. Local SMb near the center of the cell (Fig. 4 A,b) was significantly lower than those calculated near the sarcolemma (Fig.4 A, aand c). We assumed that these variations in the intracellular oxygenation reflect the intracellular gradients as oxygen molecules diffuse from the surface into the core of the cell. We then tested this hypothesis by abolishing the oxygen flux using 2 mM NaCN. Because the suppression of oxygen flux by cyanide also eliminated extracellular gradients, of the superfusion gas was reduced until the average SMb was ∼0.5. As shown in Figs. 3 B and4 B, heterogeneities of intracellular SMb were eliminated after application of NaCN. Figure 5 summarizes radial changes in SMb in the presence of 2 mM NaCN. Data were collected from three and six individual cardiomyocytes exposed to extracellular of 3.7 and 15.0 Torr, respectively. No significant change in SMb was found along the short axis of the cell.
Figure 6 shows the relationship between extracellular and SMb measured in the presence of 2 mM NaCN. The P50 determined by simple linear regression was 3.1 Torr (Fig. 6, line A), although the Hill parameter was not equal to 1. Because of the absence of heme-heme interaction in myoglobin, the Hill parameter is expected to be unity. Hence, we recalculated the regression line while fixing the Hill parameter to 1 in order to see the effect of errors in the measurement. We found a small decrement of the regression coefficient from 0.899 to 0.766 and a slight increase in the P50 from 3.1 to 3.7 Torr (Fig.6, line B).
In all cells in which the image processing was successful, a significant drop of SMb was demonstrated near the center of the cell (Figs. 1 and7). The change in SMb was then approximated by a hyperbolic curve (Figs. 1 and 7, solid curves) and subsequently converted to using the calibration data described above. We found quite steep gradients of the intracellular in the vicinity of the sarcolemma, whereas near the center of the cell was flat (Fig. 7). The lowest SMb estimated by this technique was 0.36 ± 0.07 (n = 8), which was unrelated to the size of the cell.
V˙o 2 of the quiescent cardiomyocytes at 27°C was 26 ± 9 nmol O2 ⋅ min−1 ⋅ 106cells−1(n = 5) in the presence of 1 mM extracellular Ca2+. Incubation with 1 μM CCCP increased V˙o 2to 225 ± 38 nmol O2 ⋅ min−1 ⋅ 106cells−1(n = 7) in the absence of extracellular Ca2+. These values (after temperature is compensated for) are in good agreement with theV˙o 2 of isolated rat cardiomyocytes reported by Wittenberg and Robinson (23). Thus the CCCP-treated cells in the present study represent metabolic oxygen demand of the beating rat heart at increasing (not maximal) work output (23).
In the previous study (21), we showed that average cytosolic of a single quiescent cardiomyocyte can be quantitated from the measurement of fractional oxygen binding to myoglobin using three-wavelength spectrophotometry. Because light absorption of a single cell is extremely small, we needed to carry out the spectrophotometry at the Soret band of myoglobin, where the myoglobin light absorption is maximum. This requirement, however, elicited a problem in that a significant part of the measured light absorption contains that of cytochromes because light absorption peaks of myoglobin and cytochromes significantly overlap in the Soret band. Although we have demonstrated that the estimation of average light absorption may not be seriously affected by cytochromes (see Fig. 5 of Ref. 21), the calculation of myoglobin light absorption at subcellular resolution was subject to potential errors. Moreover, the previous method did not allow us direct estimation of SMb without knowledge of molar extinction coefficients of oxy- and deoxymyoglobin in vivo. In contrast, the present technique with the use of just one wavelength enables much more accurate estimation of SMb. It is theoretically correct that light absorptions of cytochromes, even if they are large in magnitude, can be completely subtracted in the calculation of absolute SMb values (Eq. 4 ). Even values for molar extinction coefficients are not required. Another benefit of using a single wavelength is that the effect of light scattering, which strongly depends on the wavelength, can be minimized. Thus precise high spatial resolution measurement of SMb was possible in the present study.
Prerequisites for the present single-wavelength spectrophotometry were three cell images taken at three different myoglobin oxygenation states with constant cytochrome light absorptions. To fulfill these requirements, the cell was treated with NaCN or CCCP to fix the cytochromes to the respective reduced states without regard to the oxygen level. Practical issues that we encountered were the slight movements of the cell during measurement at three different values. Correction for the movement with the use of a mathematical method (affine transform) is very intricate and may not always be effective, particularly when the deflection of the cell is >5 pixels. Another methodological problem is that the current technique does not measure SMb of various intracellular points on the same cross-sectional plane of a myocyte. Instead, the calculated SMb is the volume average along the light path including SMb of the well-oxygenated sarcolemmal portion, the less oxygenated cell core, and another well-oxygenated sarcolemmal portion. Thus radial changes in SMb can in fact be detected, but the calculated desaturation at the center of the cell should be underestimated.
Radial changes in intracellular have been extensively studied theoretically and experimentally. Recent model analyses basically utilized the classic Krogh model of oxygen diffusion that was supplemented with the model of myoglobin-facilitated diffusion of oxygen (4, 8, 9, 15, 17). These analyses predicted, at least qualitatively, quite similar radial intracellular profiles. The study by Groebe (8) provides us with the most up-to-date knowledge about the model analysis. He predicted quite steep gradient at ∼10 Torr/μm in the extracellular carrier-free region in red skeletal muscle at nearly maximum performance. In contrast, the rate of fall of quickly decreases as oxygen diffuses from the sarcolemma into the cell. Facilitation of oxygen diffusion by myoglobin increases as decreases where maximum facilitation occurs at a of ∼0. Therefore, as oxygen migrates toward the cell core, a progressive increase in the fraction of deoxymyoglobin resulting from local oxygen consumption maintains oxygen flux with a smaller gradient. In addition, oxygen flux density (oxygen flux per unit area) also decreases as oxygen molecules diffuse into the cell. As a net result, gradients become remarkably shallow near the center of the cell. Finally, the intracellular a few micrometers away from the sarcolemma becomes quite low but relatively uniform in the rest of intracellular space.
Gayeski and Honig (7) conducted measurements of SMb by cryospectrophotometry in dog gracilis muscles in situ. Using a sampling volume of ∼30 μm3, they mapped SMb profiles of a cross section of single muscle cell during twitch contraction at maximalV˙o 2. They found very low but relatively uniform SMb within a cell that could be accounted for by the mathematical model of oxygen diffusion. Gayeski and Honig (5) subsequently extended their in vivo cryospectrophotometry in cardiac muscle cells of various animals. They again demonstrated quite low SMb(∼P50 of myoglobin) in the beating heart at normal work rate. Their high spatial resolution measurement of SMb showed negligible radial intracellular gradients from within 2 μm of the sarcolemma to the center of a 16-μm-diameter cell. However, these investigators did not address the steep gradients near sarcolemma that are usually predicted by model studies (4, 8, 9).
In the present study, we have directly visualized the profile of intracellular with a radial spatial resolution of 4 μm in a single cardiomyocyte. The use of a single, isolated cardiomyocyte gave us an opportunity to specifically examine diffusional oxygen transport within a cell in a situation free from the confounding effects of capillary oxygen transport. We adjusted the extracellular so that cytosolic myoglobin was partially deoxygenated as in in vivo cardiomyocytes (5). When V˙o 2of the cell was increased approximately eightfold, radial gradients of SMb, with the nadir located at the center of the cell, were clearly visualized (Fig. 2). When the measured SMb was converted to intracellular , we found a quite steep drop near the sarcolemma and relatively constant around the center of the cell (Figs. 1 and 7). These results are in good agreement with the previous theoretical studies.
We carried out these measurements at 27°C. At 37°C, on the other hand, the effective oxygen conductivity would be only slightly (∼10%)1higher (8), whereas V˙o 2 would increase by ∼1.8 times (19). Because the gradient is determined by dividing the oxygen flux density by the effective oxygen conductivity, the magnitude of intracellular gradients in vivo (37°C) would be much larger than those demonstrated at 27°C. Therefore, the dependency of the observed gradients on oxygen flux (Fig. 4) suggests the importance of interplay between intracellular oxygen diffusion resistance and oxygen flux as one of the determinants of oxygen transport to mitochondria in vivo.
As demonstrated in the present study, an increase in cellular respiration may produce large gradients, particularly in the vicinity of the sarcolemma. It is presumable that these large gradients of tend to produce regions away from the sarcolemma where oxidative phosphorylation is compromised due to the relative deficiency of diffusional oxygen supply (hypoxic core) (1). The presence of a hypoxic core would be prominent in relatively large and metabolically active cells such as cardiomyocytes. In addition, the significance of a hypoxic core may be more important in some pathophysiological conditions such as cardiac hypertrophy. At average SMb comparable to that in vivo, we demonstrated an almost flat profile near the core of a CCCP-treated single cardiomyocyte (Figs. 1 and 7). Because the rate of fall of along the diffusion path is a function of local oxygen consumption (not strictly in proportion to local V˙o 2 due to myoglobin-facilitated oxygen diffusion), the observed profile near the center of the cell seems to suggest the presence of hypoxic depression ofV˙o 2 (i.e., a hypoxic core) in a single individual cardiomyocyte.
Previous animal and model studies have addressed the major importance of myoglobin-facilitated diffusion in maintaining intracellular space at low but stable oxygenation (10, 26). Among these studies, the model study conducted by Groebe (8) isolated the effect of a hypoxic core from the effect of myoglobin-facilitated diffusion on the regulation of intracellular . He compared intracellular values calculated from the two different models. In one model,V˙o 2 was constant irrespective of , whereas in the other model, local V˙o 2 was changed as a function of the local , assuming Michaelis-Menten kinetics. Surprisingly, two types of localV˙o 2 control schema, -independentV˙o 2 versus -dependentV˙o 2, showed virtually identical radial profiles. This was because and gradients were already extremely low (<0.5 Torr, critical mitochondrial ) at the region whereV˙o 2 started to decrease (>15 μm into the fiber) andV˙o 2-dependent changes in , if any, were too small to be detected. These results, however, may not exclude the physiological importance of the hypoxic core. The mathematical model by Groebe does not include the gradients between the cytosol and the mitochondrial membranes. Although these gradients are usually believed to be very small, they may increase up to 2 Torr in cardiomyocytes when mitochondrial respiration is maximally stimulated (13, 24). Presumably, the effect of hypoxic depression ofV˙o 2 on reducing gradient (20) could be demonstrated at higher cytosolic if cytosol-mitochondrial gradients were considered. Furthermore, mitochondrialV˙o 2 becomes dependent on oxygen supply at an extremely low range (Michaelis-Menten constant = ∼0.05–0.1 Torr, see Ref. 11). Hence, a reduction of gradient, albeit very small in absolute magnitude, caused by the hypoxic reduction of localV˙o 2 would significantly affect local oxidative phosphorylation.
Cytosolic myoglobin facilitates intracellular oxygen diffusion approximately sixfold in red skeletal muscles (∼4-fold in cardiomyocytes, assuming a myoglobin content of cardiac tissue of 0.2 mM) at a of ∼0 (8). In the cardiac cell, oxygen molecules must diffuse over a distance ∼5–15 times longer in the intracellular space compared with that in the extracellular carrier-free region, until it reaches the center of the cell. Whether the fourfold increase in oxygen diffusion capability and a gradual reduction of oxygen flux in the intracellular space can fully account for the remarkably small intracellular gradients or whether depression of local V˙o 2 within a cell needs to be considered remains to be explored.
In contrast to the previous studies in red skeletal muscles at nearly maximum V˙o 2, we demonstrated that even a moderate increase in the cellularV˙o 2 results in a significant drop of intracellular near the sarcolemma. Similarly, Gayeski and Honig (5) suggested the presence of large gradients (presumably located in the extracellular carrier-free region) in the heart even at the resting metabolic oxygen demand. Current mathematical models of oxygen transport in skeletal muscles do not appear to be fully compatible with the data obtained from the cardiac cell (8). In addition, several studies demonstrated that intracellular is remarkably stable against changes in arterial oxygenation or cardiac work (5, 12). These results seem to suggest the presence of an active regulatory mechanism of intracellular oxygen transport in the heart other than simple diffusion. Such regulation may possibly be coupled with local cellular oxygen demand/metabolism (2, 22).
In conclusion, we have demonstrated in a single individual cardiomyocyte that large radial gradients may be generated within an actively respiring cell. As a result, oxidative metabolism may be partially suppressed at the core of the cardiac cell even if cellular oxygenation averaged over the cell is adequate.
We are grateful to Hiroko Tadaura (Nursing Dept. student) for technical assistance.
Address for reprint requests: E. Takahashi, Dept. of Physiology, Yamagata Univ. School of Medicine, Yamagata 990–9585, Japan.
This study was supported in part by a grant-in-aid (no. 09670037) for scientific research from the Ministry of Education, Science, and Culture of Japan and a research grant provided by the Kowa Life Science Foundation.
↵1 The effective conductivity [K o 2( )] involves the diffusion of free and myoglobin-bound oxygen. That is,K o 2( ) = αo 2 ⋅ D o 2+D Mb ⋅ CMb ⋅ P50/( + P50)2, where αo 2, CMb,D o 2, and D Mb represent O2 solubility, myoglobin concentration in tissue, and diffusion coefficientsfor oxygen and myoglobin molecules, respectively. Papadopoulos et al. (16) reported that Q10 forD Mb for skeletal muscle cell is 1.5, whereas that for αo 2 ⋅ D o 2is 1.15 (26). With the use of the values for these parameters depicted by Groebe (see Table 1 of Ref. 8), maximumK o 2at 37°C would be 1.09 times larger than that at 27°C.
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