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1Department of Physiology II, Nara Medical University, Kashihara 634-8521; and 2Department of Anatomy, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan
Submitted 13 November 2003 ; accepted in final form 19 February 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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mVO2 (the mMVO2 with stimulation – the mMVO2 without stimulation) was 0.83 ± 0.12 ml O2·min–1·100 g LV–1. There were no differences between mean mMVO2 with and without stimulation in Ca2+-free solution. The increases in extracellular Ca2+ concentrations up to 14.4 mM did not affect the mMVO2 without stimulation but significantly increased the mMVO2 with stimulation up to 140% of control. The mMVO2 significantly increased up to 190% of the control in a dose-dependent manner. In contrast, the shortening did not increase in a dose-dependent manner. Cyclopiazonic acid (CPA; 30 µM) significantly reduced the mMVO2 to 0.27 ± 0.06 ml O2·min–1·100 g LV–1 (35% of control). The combination of 5 mM 2,3-butanedione monoxime (BDM) and 30 µM CPA did not further decrease mMVO2. Although BDM (3–5 mM) decreased the mMVO2 by 28–30% of control in a dose-independent manner, 3–5 mM BDM decreased shortening in a dose-dependent manner. Our results indicate that the mMVO2 of mouse LV slices during shortening under mechanically unloaded conditions consists of energy expenditure for total Ca2+ handling during excitation-contraction coupling, basal metabolism, but no residual cross-bridge cycling.
excitation-contraction coupling; basal metabolism; free shortening
Recently, genetic manipulation of cardiac-specific genes in transgenic and knockout murine models holds promise for studies focused on exploring the specific role of contractile elements and Ca2+-handling constituents in E-C coupling (21, 22, 26). To interpret findings in such genetically manipulated mice, evaluation of cardiac function in the normal mouse should be accomplished in terms of the coupling of cardiac mechanical work and energetics because there are significant differences between rats and mice (12). Nevertheless, there are only a few reports on mouse cardiac mechanical work (4, 5, 8, 16) and energetics (14).
In 1998, Kameyama et al. (14) completed an analysis of cardiac mechanical work and energetics in mice. They tested the feasibility of an isolated, balloon-in-ventricle, isovolumically contracting, crystalloid-perfused mouse heart preparation for studies of cardiac mechanoenergetics using the end-systolic pressure-volume relation (ESPVR) and MVO2 per beat and pressure-volume area (PVA) framework employed in larger species (15, 27, 28, 30). The MVO2-PVA relation was well fitted by a straight line and the value of MVO2 at 0 PVA (MVO2 intercept value) corresponding to mechanically minimally unloaded MVO2 in mouse LV was derived (14). The MVO2 intercept value includes MVO2 for total Ca2+ handling in E-C coupling and for basal metabolism in dogs and rats (6, 7, 9, 27, 29). The MVO2 intercept value, however, is threefold of that in the rat LV (10, 25) and thus residual mechanical loading-derived MVO2 may be included.
The aim of the present study was to test the feasibility of a system using mouse LV myocardial slices of 300-µm thick for studies of cardiac energetics, especially the measurement of basal mMVO2 and mMVO2 for total Ca2+ handling in E-C coupling using oximetry employed for rat LV 300-µm-thick slices.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The investigation conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996).
Adult male Crj:CD-1 (ICR) mice (10–14 wk, 30–50 g; Charles River Japan; Yokohama, Japan) were anesthetized with pentobarbital sodium (50 mg/kg ip). A tracheotomy was performed, and the mouse was given 100% O2 through a respirator. The chest was opened midsternally, and heparin sodium (1,000 U/kg ip) was then injected. A 23-gauge stainless steel cannula was inserted from the aortic arch down to just above the aortic valve with the use of a cannula-guiding apparatus. This cannula was then connected to a modified Langendorff perfusion system to completely wash out the coronary blood at a perfusion pressure of 80 cmH2O. The whole heart was excised under the perfusion with Tyrode solution oxygenated with 100% O2 at 12°C for 5 min. The composition of Tyrode solution (in mmol/l) was 136.0 NaCl, 5.4 KCl, 1.0 MgCl2·6H2O, 0.33 NaH2PO4·2H2O, 1.8 CaCl2·2H2O, 10.0 glucose, and 5.0 HEPES, with pH adjusted to 7.4 with NaOH at 30°C.
After perfusion, both atria, the four valves, including connective tissue, the aorta, and the pulmonary artery were removed from the heart. With the use of a microslicer (model DTK-3000, Dosaka; Kyoto, Japan), each whole LV was cut into 300-µm-thick slices along the LV long axis in parallel with the septum. We obtained 12–16 slices from two mice (single-side surface area, 
23.0 ± 7.5 mm2; length and width, 4.8 ± 1.8 and 5.2 ± 1.9 mm, respectively). The slices were stored in Tyrode solution oxygenated with 100% O2 at 18°C for 30 min and subsequently at 25°C at least for 30 min. Finally, the mMVO2 of the slices was measured 2.5 h after excision of the heart.
Assessment of mMVO2 of Myocardial Slices
A set of 1216 myocardial slices was used to measure mMVO2 of slices in the absence and presence of electrical field stimulation. Stimulation consisted of 1-Hz rectangular pulses, 0.5 ms in duration, and current of 0.8 A. The direction of stimulation was alternated by a polarity change. We did not find the bubbles of oxygen and hydrogen on the electrode under a microscope. mMVO2 of a set of slices was measured polarographically with an oxygen electrode (model 1T-125, Instech Labs; Plymouth Meeting, PA) and current amplifier (model 102, Instech Labs), as described previously (17, 18, 29, 31). This oximetric system was calibrated at three different O2 concentrations (in mg/l): 0 (5% Na2SO3 solution), 7.3 (Tyrode solution saturated with air), and 35.1 (Tyrode solution saturated with 100% O2) at 30°C in each experiment. Volume of Tyrode solution in the chamber was measured at the end of the three repeated measurements.
We first measured the background mM
O2 (0.19 ± 0.07 mg O2/min; n = 72 experiments) without myocardial slices in the Tyrode-filled chamber at the beginning and end of each measurement. The wet weight of a set of slices was 81–146 mg. We obtained mMVO2 of slices by subtracting the background mMVO2 (A: mg O2/min) from the measured mMVO2 (B: mg O2/min) in the presence of myocardial slices. The mMVO2 (ml O2·min–1·100 g LV–1) was calculated as [(B – A)/slice wet weight (in grams)] x volume of Tyrode solution in the chamber (in liters) x [22.4 (liters)/32 (g)] x 100 (g). MVO2 was indicated as µl O2·beat–1·g LV–1. Oxygen concentration data were acquired by a 12-bit analog-to-digital converter at a sampling rate of 2 Hz [model AD12-16 (PCI) E; Contec] and displayed on a desktop computer (model FM-V, Fujitsu) with the use of software developed in Active-X (Contec) that was written in Microsoft Visual Basic. The slope of A or B was determined by regression analysis in 120-s periods during nonstimulation (2 min) and in the last 80-s periods during stimulation (3 min).
Image Analysis of Motility of Mouse Myocardial Slice
A piece of slice was placed in a 2.58 ± 0.09-ml oximetry chamber (n = 72 experiments). The slice was gently superfused with Tyrode solution for 10 min, during which the magnetic stirrer was rotated. During image analysis, the airtight chamber was closed and the stirrer was turned off. Slice contractions under mechanically unloaded conditions were evoked by the same 1-Hz field stimulation as used in the oximetry.
We monitored and recorded the video images of signals discriminated by a selected area (5 mm x 10 mm) containing slice image during slice contraction at video rates with a microscopic-video area analyzer (model C3163, Hamamatsu Photonics; Shizuoka, Japan) and a SZH-131 DA color charge-coupled device camera (model CS 220, Olympus; Tokyo, Japan). The area analyzer and camera were controlled and programmed by a NEC computer (model PC-98) using N88-BASIC with a National Instruments GPIB interface and a videocassette recorder (model HR-D 380; Victor; Tokyo, Japan). We evaluated the slice motility as the relative change (%motility index) in the total number of pixels of the discriminated video signal of slice images in contraction and relaxation cycles, as proposed by Takaki et al. (29) in 1998. We averaged the motility index of intermittent seven contraction-relaxation cycles at 20-s intervals throughout the latter 2 min of stimulation period (3 min).
Experimental Procedure
Tyrode solutions were oxygenated with 100% O2 and preheated to 30°C in a water bath. Slices were placed into the oximetric chamber filled with the prepared Tyrode solution, which was continuously oxygenated with 100% O2. The chamber was warmed with the use of a Microwarm plate (model DC-MP10DM, Kitazato; Tokyo, Japan). After the slices were superfused for 10 min, the chamber was closed tightly with a transparent acrylic lid by four screws to prevent any leak of water or O2. mMVO2 of the quiescent slices without stimulation [St(–) mMVO2]was obtained for 120-s of nonstimulation period (2 min), and mMVO2 of the activated and contracting slices with stimulation [St(+) mMVO2] was obtained for the last 80 s of the stimulation period (3 min). The slices in the chamber were then washed for 10 min with a new Tyrode solution, saturated with 100% O2, for 10 min.
Experimental Protocol
We used the following protocols for studies 1–5.
Protocol 1. After 25-min superfusion of the slices with normal Tyrode (NT) solution (1.8 mM Ca2+), the first St(–) mMVO2 and St(+) mMVO2 measurements (the first measurement) were performed as described above. Subsequently, the second and third measurements of St(–) mMVO2 and St(+) mMVO2 were performed in NT (NT series). Thus the measurements were repeated three times to ascertain the reproducibility of measurements.
Protocol 2. After all of the procedures were performed with the use of nominally Ca2+-free Tyrode solution instead of NT, St(–) mMVO2 and St(+) mMVO2 measurements were performed (as described in protocol 1) after final 25-min superfusion of the slices with nominally Ca2+-free Tyrode solution.
Protocol 3. For two [Ca2+] (3.6 mM) series, after the first measurement in NT, the slices were superfused for 10 min with 3.6 mM Ca2+ Tyrode solution and the second measurement in 3.6 mM Ca2+ Tyrode solution was performed. Again, the slices were superfused for 10 min with NT and the third measurement in NT was performed. Ca2+ (7.2 mM and 14.4 mM), BDM (3–5 mM), CPA (10–30 µM), and DMSO (0.15%) series were performed as in this manner. In CPA and DMSO series, field stimulation was performed for the former 5 min during the 10-min superfusion with CPA or DMSO.
Study 1.
This study used mechanically unloaded mMVO2 (Fig. 1A) and motility index of slices (Fig. 1B) in NT. The increment in mMVO2 by stimulation was obtained as mMVO2 = St(+) mMVO2 – St(–) mMVO2.
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mMVO2, and motility after the first measurements in NT.
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mMVO2, and motility of slices at 10 min after the first measurements of mMVO2 in NT (Fig. 3). In addition, we examined the effects of CPA (30 µM) plus BDM (5 mM) on St(–) mMVO2 and mMVO2 at 10 min after the first measurements of mMVO2 in NT.
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mMVO2, and motility at 10 min after the first measurements in NT.
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The viability of the myocardial cells and the influence of procedures in three repeated measurements on the viability were examined by light microscopic observation. The myocardial slices were fixed in Zamboni's solution before and after mMVO2 measurements. The samples were routinely processed, embedded in hydrophilic resin (Technovit 7100, Kulzer; Friedrichsdorf, Germany), and stained by 0.1% Toluidine blue. Two-micrometer cross sections were obtained. Each section (n = 9) arbitrarily selected from five myocardial slices was made to have the cross sections of the bilateral surface of the slice. On microscopic examination, morphologically intact cell number (Ni) and damaged cell number (Nd) was counted along a single arbitrary chosen vertical line across each section. Intact cell ratio was calculated as Ni/(Ni + Nd), as previously reported (31).
Ultrastructual Observation
For electron microscopic examination, the same myocardial slices used for mMVO2 measurement were fixed by immersion in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. We removed the bilateral surface layers and used the intact core of a slice. The core of the slice was trimmed into small pieces (1 x 1 mm). The tissue pieces were immersed in the same fresh fixative and fixed further at 4° for 4 h. After being rinsed with buffer, the specimens were postfixed for 3 h with a 1.0% OsO4 solution in 0.1 M phosphate buffer, pH 7.4, dehydrated through an ascending series of ethanol, and substituted with propylene oxide and embedded in epoxy resin. Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and observed with an electron microscope (model JEM1200 EXII; JEOL; Tokyo, Japan).
Statistics
All data are presented as means ± SD. Multiple comparisons were performed by one-way and repeated-measures ANOVA and Bonferroni t-test. In all statistical tests, P values < 0.01 or 0.05 were considered statistically significant.
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RESULTS |
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Figure 1A shows mMVO2 of mechanically unloaded LV myocardial slices (n = 12–16 each) without [St(–) mMVO2] and with stimulation [St(+) mMVO2] in NT at first, second, and third measurements in 8 sets of slices from 16 hearts. The St(–) mMVO2 of the slices was 0.97 ± 0.14 ml O2·min–1·100 g LV–1 (3.2 mW/g) at second measurement. The St(+) mMVO2 of the same set of slices was significantly increased to 1.80 ± 0.17 ml O2·min–1·100 g LV–1 (0.30 µl O2·beat–1·g LV–1) (P < 0.001). mMVO2 was 0.83 ± 0.12 ml O2·min–1·100 g–1 and was 86% of St(–) mMVO2. St(–) mMVO2 and mMVO2 also were fairly constant among three repeated measurements (P > 0.05).
On the other hand, there was no significant difference between mechanically unloaded St(–) mMVO2 and St(+) mMVO2 in Ca2+-free Tyrode solution in another group of nine paired experiments (Fig. 2A).
Ca2+ Dependency
Figure 2B shows St(–) mMVO2 and mMVO2 in four groups of 6–8 sets of slices at various extracellular Ca2+ concentrations. There were no significant differences among mean St(–) mMVO2 values at increased Ca2+ concentrations from 1.8 mM Ca2+ (NT) to 14.4 mM Ca2+. Mean mMVO2 increased up to 190% of that in 1.8 mM Ca2+ in an extracellular Ca2+ concentration-dependent manner. Although mean mMVO2 at 3.6 mM Ca2+ (n = 8 sets) did not increase significantly from that in 1.8 mM Ca2+, mean mMVO2 significantly (P < 0.01) increased at 7.2 mM Ca2+ (n = 8 sets) and 14.4 mM Ca2+ (n = 6 sets) from that in 1.8 mM Ca2+ (n = 8 sets) and significantly (P < 0.01) increased from that at 7.2 mM Ca2+ to 14.4 mM Ca2+. We occasionally observed arrythmias at 14.4 mM Ca2+, although in few slices. Thus we did not examine St(–) mMVO2 and mMVO2 at Ca2+ concentrations >14.4 mM Ca2+.
Effects of CPA
Figure 3 shows mean St(–) mMVO2 and mMVO2 in 4 groups of 6–8 sets of slices in NT (n = 8 sets), DMSO (n =6 sets), and 10 µM CPA (n = 7 sets) and 30 µM (n = 6 sets). There were no significant differences in St(–) mMVO2 among NT, DMSO, and CPA (10 and 30 µM). In contrast, mean mMVO2 in CPA 10–30 µM significantly (P < 0.001) decreased to 48–35% of that in NT in a dose-dependent manner. The combination of 5 mM BDM and 30 µM CPA did not further decrease mMVO2 (0.23 ± 0.11 ml O2·min–1·100 g LV–1; 35% of that in NT; n = 6).
Effects of BDM
We determined the effective concentration of BDM to be 3–5 mM because we have previously confirmed specific inhibitory effect on cross-bridge cycling of 5 mM BDM without any changes in St(–) mMVO2 and St(+) mMVO2 in rat mechanically unloaded LV myocardial slices (17, 31). Figure 4 shows mean St(–) mMVO2 and mMVO2 in 3 groups of 6–8 sets of slices in NT (n = 8 sets) and 3 mM (n = 8 sets) and 5 mM (n = 6 sets) BDM. There were no significant differences in St(–) mMVO2 among NT, and BDM (3 and 5 mM). In contrast, mean mMVO2 in BDM at the same doses significantly (P < 0.05) decreased by 28–35% of that in NT, although not dose dependently.
Mechanically Unloaded Contractions of Mouse Myocardial Slice
Figure 1B shows relative changes (% of first trial) of motility index of unloaded LV myocardial slices with stimulation in NT at first, second, and third trials in 10 slices from 4 hearts. Mean motility index values were 2.18 ± 0.77% in a first trial. Relative changes of motility index were fairly constant among the three repeated measurements (P > 0.05).
Figure 5 shows each series of 10 contraction and relaxation cycles in control (Fig. 5, A and C) and in 3–5 mM BDM (Fig. 5, B and D). Figure 5, A–D, shows the same sets of slices. In each series in control, free shortenings were fairy constant. BDM moderately decreased the intensity of the free shortening at 3 mM and abolished the free shortening at 5 mM.
Figure 6 shows relative changes of motility index in NT (99.2 ± 7.6% of first trial in NT; n = 10 slices) and 3 mM BDM (53.7 ± 21.8% of first trial in NT; n = 10 slices) and 5 mM BDM (15.8 ± 15.9% of first trial in NT; n = 8 slices) groups. Relative changes of motility index were dose dependently decreased by BDM from that in NT, and there were significant differences between NT and BDM (3 and 5 mM; P < 0.001). After washout of BDM, we observed an almost full recovery from the decreased motility index (P > 0.05). CPA (30 µM) decreased motility index to 25.8 ± 18.8% (n = 8) in four hearts (data not shown).
Figure 7 shows each series of 10 contraction and relaxation cycles in control (Fig. 7, A, C, and E) and in (in mM) 3.6 Ca2+, 7.2 Ca2+, and 14.4 Ca2+ (Fig. 7, B, D, and F). Figure 7, A–F, shows the same sets of slices. In each series in control, free shortenings were fairy constant. At 3.6 mM, Ca2+ markedly increased the intensity of the free shortening (Fig. 7B), but 7.2 mM Ca2+ and 14.4 mM Ca2+ did not further increase the intensity of the free shortening (Fig. 7, D and F). Figure 8 shows relative changes of motility index in 1.8 mM Ca2+ (NT) (99.2 ± 7.6% of first trial in NT; n = 10 slices) and 3.6 mM Ca2+ (170.9 ± 54.1% of first trial in NT; n = 12 slices) and 7.2 mM Ca2+ (176.3 ± 29.2% of first trial in NT; n = 12 slices) and 14.4 mM Ca2+ (168.1 ± 42.2% of first trial in NT; n = 12 slices) groups. Relative changes of motility index significantly increased from 1.8 mM Ca2+ by 3.6–14.4 mM Ca2+ (P < 0.001), but there were no significant differences among 3.6–14.4 mM Ca2+ (P > 0.05). After washout of 3.6 mM Ca2+, we observed almost full recovery from the increased motility index, but not after washout of 7.2–14.4 mM Ca2+ (data not shown).
Light Microscopic Observations
By light microscopy, the vast majority of myocardial cells and the small number of Purkinje fiber showed normal morphology except for the cells interfacing the surfaces of each slice. Myocardial cells aligned in the slice surface were swollen, showed a low staining in cytoplasm, and had a ghost cell-like appearance. The mean intact cell ratios were 83.9 ± 3.2% and 76.4 ± 8.2% before and after mMVO2 measurements (n = 9 slices x 1 site = 9 each). The difference was not statistically significant (P > 0.05).
Ultrastructual Observations
Figure 9 shows electron micrographs of myocardial slices before and after mMVO2 measurements. Most myocardial cells in the middle layer of the slices showed neither ultrastructual changes, such as swelling and fusion of mitochondria, nor irreversible necrotic changes, such as the appearance of dense deposits even after mMVO2 measurements. These ultrastructural observations indicate that most myocardial cells were kept intact throughout the measurements.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The second finding was that main part of mMVO2 [St(+) mMVO2 – St(–) mMVO2] is energy expenditure utilized for total Ca2+ handling in EC coupling in mouse LV slices as in rat LV slices (29, 31). This finding is evidenced by 1) mMVO2 was increased in a Ca2+ concentration-dependent manner and by 2) mMVO2 was decreased by CPA in a dose-dependent manner.
However, in mouse LV slices, unlike in rat LV slices (29, 31), 3–5 mM BDM significantly decreased mMVO2 by 28–30%. Furthermore, after 30 µM CPA treatment, 35% of mMVO2 remained. Therefore, we assumed that mMVO2 might include energy expenditure utilized partly for residual cross-bridge cycling. If 5 mM BDM directly inhibited cross-bridge cycling, the combination of 30 µM CPA and 5 mM BDM would further decrease mMVO2. However, the combination did not decrease further mMVO2, suggesting that the BDM-mediated decrease in mMVO2 was not mediated by directly inhibiting cross-bridge cycling. The inhibitory effect of CPA on mMVO2 for Ca2+ handling in E-C coupling is maximum at 30 µM because CPA did not decrease further mMVO2 at 100 µM (our unpublished observation). For this reason, the inhibitory effect of 5 mM BDM on mMVO2 for Ca2+ handling must be masked. Consequently, in mouse slices as in rat myocardial slices, the possibility that mMVO2 for the residual cross-bridge cycling is negligibly small and the BDM-induced decrease in mMVO2 is due to inhibition of Ca2+ handling in E-C coupling is probable. In fact, there are many reports showing that BDM decreases energy expenditure utilized for total Ca2+ handling in E-C coupling (1, 11, 20, 24).
As shown in Figs. 6 and 8, the effects of BDM and extracellular Ca2+ on mMVO2 did not correlate with those on motility index. The effect of BDM on motility index was dose dependent, but that on mMVO2 was not dose dependent, i.e., definite (28–30%). Conversely, the effect of extracellular Ca2+ on mMVO2 was dose dependent, but the effect on motility index was not dose dependent. Motility index indicates only residual cross-bridge cycling at mechanically unloaded state and thus motility is originally small. Therefore, we supposed that even at a lower Ca2+ concentration (3.6 mM), motility index reached maximum value. On the other hand, mMVO2 mainly includes E-C coupling MVO2 (at least 65% of mMVO2; see Fig. 3). E-C coupling MVO2 must increase in a [Ca2+]-dependent manner. At 7.2 mM, no arrhythmia was observed and even at 14.4 mM arrhythmia was seldom in slices, suggesting that slices are normal. This might be partly due to the low-frequency (1 Hz) stimulus compared with highly frequent beating of in vivo hearts (400–600 beats/min). Taken together, we think that a substantial fraction of the cells on the surfaces of the slices is not damaged.
Basal mMVO2 that we observed in mice LV slices were unexpectedly smaller than those in rat LV myocardial slices (0.97 vs. 2.09 ml O2·min–1·100 g–1), but mMVO2 in mice LV slices (0.83 ml O2·min–1·100 g–1) did not differ from that in rat LV slices (0.91 ml O2·min–1·100 g–1) (see Ref. 29). Basal mMVO2 is believed to correlate reversely with body weight (19). It is unclear why basal mMVO2 is smaller in mouse LV slices than that in rat LV slices. Gibbs and Loiselle (6) suggested the possibility that time-dependent exponential declines in basal metabolism exist in many hearts. Mouse and rat MVO2 is measured 2.5 h after excision of the heart. Therefore, even if the initial value of mouse slices were larger, the basal metabolism in mouse slices would be smaller than in rat slices due to time-dependent exponential decline if the initial decline speed in mouse slices is faster than in rat slices.
Basal mMVO2 slightly declined with time in mouse LV slices as in other preparations (6), although the effects of Ca2+, BDM, and CPA were tested at the second measurement after the first measurement in NT to compare the same second measurement in NT.
The MVO2-PVA relation was well fitted by a straight line and the value of MVO2 at 0 PVA (MVO2 intercept value) corresponding to mechanically minimally unloaded MVO2 in the mouse LV of whole heart preparation has been reported (14). The MVO2 intercept value theoretically corresponds to St(+) mMVO2 in mice LV myocardial slices in the present study. The MVO2 intercept value (0.92 µl O2·beat–1·g LV–1), however, is practically threefold of St(+) mMVO2 (0.30 µl O2·beat–1·g LV–1) in mice LV myocardial slices in the present study. This is quite different from the results in the rat heart. The MVO2 intercept value in the rat whole heart preparation is practically identical to St(+) mMVO2 in rat LV myocardial slices (10, 25, 29, 31). Although this is partly due to the difference of temperature (30°C vs. 35–37°C), it seems unlikely that the difference of used Ca2+ concentration (1.8 vs. 2.5 mM) is the cause of the difference between MVO2 intercept value and St(+) mMVO2 because there were no significant differences in St(+) mMVO2 at 1.8 and 3.6 mM (2 Ca2+) in the present study. The measurements made by Kameyama et al. (14) may be an overestimate due to the loss of oxygen across the epicardial surface of the perfused heart. Another overestimate may be due to energy expenditure for residual cross-bridge cycling. This might be possible because it is difficult to make completely mechanical unloading conditions in the mouse whole heart preparation differently from the rat whole heart preparations (10, 25).
Limitation
In the present study, we observed 2.2% of motility index for mice LV slices, whereas free shortening of mouse cardiac cells are 5–10% (13). The relatively small motility index seems to be partly due to the heterogeneity of muscle fiber orientations of mouse LV slices as in rat LV slices (29). Nevertheless, the relative changes of motility index could be beneficial to evaluate the effects of agents acting on residual cross-bridge cycling.
In conclusion, the present study indicated that mMVO2 of 300-µm-thick slices from mouse LV under quiescent condition represented basal mMVO2 used for cardiac basal metabolism and that the increase in mMVO2 of the slices with 1-Hz field stimulation mainly represented energy expenditure used for total Ca2+ handling in E-C coupling in the heart.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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Present address of H. Kohzuki: Dept. of Food and Nutrition, Okayama Gakuin University, 787 Aruki, Kurashiki 710-8511, Japan.
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FOOTNOTES |
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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.
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REFERENCES |
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O2 in isolated rabbit heart: comparison of mechanical loading and BDM method. Am J Physiol Heart Circ Physiol 273: H1032–H1037, 1997.O2 of mechanically unloaded contraction of rat ventricular slices measured by a new approach. Am J Physiol Heart Circ Physiol 270: H1063–H1070, 1996.
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P < 0.01, significantly different vs. 
