To clarify the energy-expenditure mechanism during Ba2+ contracture of mechanically unloaded rat left ventricular (LV) slices, we measured myocardial O2 consumption (V˙o 2) of quiescent slices in Ca2+-free Tyrode solution andV˙o 2 during Ba2+ contracture by substituting Ca2+ with Ba2+. We then investigated the effects of cyclopiazonic acid (CPA) and 2,3-butanedione monoxime (BDM) on the Ba2+ contractureV˙o 2. The Ca2+-freeV˙o 2 corresponds to that of basal metabolism (2.32 ± 0.53 ml O2 ⋅ min−1 ⋅ 100 g LV−1). Ba2+ increased theV˙o 2 in a dose-dependent manner (from 0.3 to 3.0 mmol/l) from 110 to 150% of basal metabolic V˙o 2. Blockade of the sarcoplasmic reticulum (SR) Ca2+ pump by CPA (10 μmol/l) did not at all decrease the Ba2+-activatedV˙o 2. BDM (5 mmol/l), which specifically inhibits cross-bridge cycling, reduced the Ba2+activatedV˙o 2 almost to basal metabolic V˙o 2. These energetic results revealed that the Ba2+-activatedV˙o 2 was used for the cross-bridge cycling but not for the Ca2+ handling by the SR Ca2+ pump.
- oxygen consumption
- excitation-contraction coupling
- sarcoplasmic reticulum calcium adenosine 5′-triphosphatase
- cyclopiazonic acid
- 2,3-butanedione monoxime
we have instituted rat left ventricular (LV) slices (300 μm thick) in a perfusion and electrical stimulation chamber as an appropriate preparation to analyze myocardial O2 consumption (V˙o 2) under mechanically unloaded conditions (29, 31, 36). We expected that these myocardial slices would be virtually free from the mechanical constraints that they had in situ. Using this new preparation, we have revealed that an increment inV˙o 2 by 1-Hz stimulation consists of V˙o 2 for Ca2+ handling in the excitation-contraction (E-C) coupling (E-C couplingV˙o 2) but not for residual cross-bridge cycling. We also have revealed thatV˙o 2 without stimulation represents a constant basal metabolism (basal metabolicV˙o 2) in both normal and Ca2+-free Tyrode solution (29,36). However, the energy expenditure due to the increase in cross-bridge cycling of these myocardial slices remains to be analyzed.
The aim of the present study was, first, to detect an increase in energy expenditure or V˙o 2 due to the increase in cross-bridge cycling by Ba2+ contracture of myocardial slices from the rat LV. Our second goal was to analyze the mechanism of the increased V˙o 2 by using a sarcoplasmic reticulum (SR) Ca2+-pump blocker, cyclopiazonic acid (CPA) (2, 29), and a cross-bridge cycling inhibitor, 2,3-butanedione monoxime (29, 36).
There is no direct evidence indicating whether Ba2+ exclusively activates the contractile system (4, 13, 17, 22, 23). Because Ba2+ contracture is induced by displacing Ca2+ with Ba2+, an incomplete washout of Ca2+ during the superfusion with nominally Ca2+-free Tyrode solution may be one possibility. Although we have previously reported that E-C coupling V˙o 2 is not detected in nominally Ca2+-free Tyrode solution, even when electrical stimulation is performed (36), the possibility that Ba2+ releases residual Ca2+ from the SR via a mechanism similar to the Ca2+-induced Ca2+-release mechanism (5, 6) should be considered. However, there is little supporting evidence for this possibility (13, 25). To the contrary, there is evidence that Ba2+ blocks SR Ca2+ release in triadic SR preparations, even though these preparations are isolated from skeletal muscle (19, 20). Although there is another possibility that Ba2+ is sequestered and released (i.e., cycled) by the SR, just as Ca2+ and Sr2+ are (26), many studies have suggested that Ba2+ is not cycled (1, 9, 14, 15, 19, 32). Displacing Ca2+ with Ba2+ can activate contractile protein interactions, but it does not support E-C coupling in skeletal muscle (9). Furthermore, Ba2+ has no effect on SR Ca2+ ATPase activity in skeletal muscle SR (14) and myocardial SR (1) and on Ca2+ uptake by skeletal muscle SR (15, 19, 32). In agreement with these studies suggesting the lack of Ba2+ cycling in SR, we obtained results of the energetics to reveal that theV˙o 2 of Ba2+-activated myocardial slices was mainly used for the cross-bridge cycling but not for the residual Ca2+ handling by the SR Ca2+ pump.
LV Myocardial Slice Preparation
Forty-one adult male crj:Wistar rats weighing 300–420 g (mean weight 358 ± 29 g) were purchased from Charles River Japan (Yokohama, Japan) and anesthetized with pentobarbital sodium (50 mg/kg ip). All experimental procedures were described previously in detail (29, 36). Briefly, the whole heart was excised after perfusion with Tyrode solution at 12°C gassed with 100% O2 for 5–10 min. The composition of Tyrode solution (in mmol/l) was 136.0 NaCl, 5.4 KCl, 1.0 MgCl2 ⋅ 6H2O, 0.33 NaH2PO4 ⋅ 2H2O, 0.9 CaCl2 ⋅ 2H2O, 10.0 glucose, and 5.0 HEPES, with pH corrected to 7.4 with NaOH at 30°C. Each myocardial slice was cut into 300-μm-thick slices in parallel with the epicardium with a microslicer (DTK-3000; Dosaka EM, Kyoto, Japan). We obtained 17–24 slices from each heart. The slices were stored for 30 min in 18°C Tyrode and then for at least 30–60 min in 25°C Ca2+-free Tyrode solution gassed with 100% O2. Ca2+-free Tyrode solution was made by excluding CaCl2 ⋅ 2H2O in normal Tyrode solution. Ba2+Tyrode solution was made by displacing CaCl2 ⋅ 2H2O with BaCl2.
Ba2+-ActivatedV˙o2 of Myocardial Slices
To obtain a steady contracture with no spontaneous twitch (23), the myocardial slices were incubated for 10 min with Ca2+-free Tyrode solution.V˙o 2 for these quiescent slices was measured for 6 min in an oximetric chamber at 30°C. The subsequent superfusion with Ba2+Tyrode solution was employed to induce Ba2+ contracture of the slices without electrical stimulation. We observed Ba2+ contracture under an inverted microscope.
V˙o 2 of a set of six myocardial slices on average was measured polarographically with an O2 electrode (1T-125; Instech Laboratories, Plymouth Meeting, PA), the head of which faced the solution in the air-tight (oximetric) chamber. The electrode was connected to a current amplifier (model 102; Instech Laboratories), and its output was recorded with a flatbed recorder (model FBR-252A; TOA Electronic, Tokyo, Japan). This oximetric system was calibrated at three different O2 concentrations [0 mg/l (5% Na2SO3solution), 7.3 mg/l (Tyrode solution saturated with air), and 35.1 mg/l (Tyrode solution saturated with 100% O2)] at 30°C in each experiment.
Assessment of V˙o2 of Myocardial Slices
We first examined the backgroundV˙o 2 with no myocardial slices in the Tyrode-filled chamber at the beginning and end [0.42 ± 0.15 and 0.39 ± 0.15 ml O2/min, respectively;n = 52 sets of 4–6 slices;P > 0.05] of each measurement of the same slices at 30°C. MyocardialV˙o 2 was obtained by subtracting the backgroundV˙o 2(A; in scales/min) from the measuredV˙o 2(B; in scales/min) with myocardial slices present in the oximetric chamber. MyocardialV˙o 2 (in ml O2 ⋅ min−1 ⋅ 100 g LV−1) was calculated as [(B −A) × 0.399/slice wet weight (in grams)] × volume of Tyrode solution in the chamber (in liters) × [22.4 liters/32 g] × 100 g (1 scale of the reading of the trace = 0.399 mg/l in the present study). We determined the wet weight of each set of slices [58.7 ± 13.5 mg; n =52 sets of slices] so that the ratio of Bto A became higher than 2.0. The maximum sensitivity of the O2measurement was obtained whenB/Awas 2.0, i.e., 0.92 μl O2/min in this chamber. We have previously confirmed that the myocardial sliceV˙o 2 was reproducible in three repeated measurements in the same protocol without stimulation at 30°C (29).
Any Tyrode solutions were gassed with 100% O2 and preheated to 30°C in a water bath. Six slices on average were placed in the air-tight chamber filled with the prepared 2.62 ± 0.04 ml (n = 52 sets of slices) Tyrode solution gassed with 100% O2. These slices were preincubated and then incubated in Ca2+-free Tyrode solution without and with Ba2+ throughout the experiment. E-C coupling V˙o 2was not detected under these conditions, even when electrical stimulation was performed (36). MyocardialV˙o 2 of Ba2+-activated slices without electrical stimulation was not different from that with stimulation. Therefore, we measured theV˙o 2 of all slices without stimulation. We used the following protocols in four different groups of slices.
There were 31 sets of slices from 20 rats in protocol 1. After 10-min incubation of a set of slices in Ca2+-free Tyrode solution, the first V˙o 2 measurement was performed for 6 min. This value was used as a control value. After the first washout of the slices with Ba2+ Tyrode solution for 10 min, the second V˙o 2 measurement of Ba2+-activated slices in this solution was performed for 6 min. The concentration of Ba2+ was increased from 0.3 (n = 6), 0.5 (n = 10), and 1 (n = 6) mmol/l to 3 mmol/l (n = 9). After theV˙o 2 of Ba2+-activated slices was measured, the slices were washed out with Ca2+-free Tyrode solution for 10 min and the V˙o 2 of the noncontracted slices was measured for 6 min. These values were used as back control values.
Eight sets of slices from eight rats were used inprotocol 2. To confirm the stability of V˙o 2 of Ba2+-activated slices, theV˙o 2 was repeatedly measured in the same set of slices after the measurement ofV˙o 2 of the slices in Ca2+-free Tyrode solution. After 10-min incubation of a set of slices in Ca2+-free Tyrode solution, the first V˙o 2 measurement was performed for 6 min. This value was used as a control value. After the washout of the slices with Ba2+Tyrode solution for 10 min, the secondV˙o 2 measurement of Ba2+-activated slices in this solution was performed for 6 min. The concentration of Ba2+ was 1 mmol/l. After theV˙o 2 of Ba2+-activated slices was measured, the activated slices were again superfused with Ba2+ Tyrode solution for 5 or 10 min and the third V˙o 2measurement was performed for 6 min. Total time of treatment with Ba2+ was at least 27–32 min.
Seven sets of slices from seven rats were used inprotocol 3, and CPA (10 μmol/l) in 1.3% dimethyl sulfoxide was used as a vehicle. Control values were obtained as in protocol 1. After washout of the slices with Ba2+ (1 mmol/l) Tyrode solution for 10 min, the secondV˙o 2 measurement of Ba2+-activated slices was performed for 6 min. This value was used as the Ba2+ (1 mmol/l)V˙o 2 value. Pretreatment of CPA (10 μmol/l) in Ca2+-free Tyrode solution for 5 min completely blocked Ba2+ contracture by Ba2+ (1 mmol/l) Tyrode solution in the presence of CPA (10 μmol/l) for 10 min (unpublished observation). Therefore, we added CPA (10 μmol/l) after the secondV˙o 2 measurement of Ba2+-activated slices as follows: after washout of the slices with Ba2+ (1 mmol/l) Tyrode solution containing CPA (10 μmol/l) for 5 min, the thirdV˙o 2 measurement in this solution was performed for 6 min. This value was used as the CPAV˙o 2 value. To confirm the recovery from activation with Ba2+(1 mmol/l) Tyrode solution containing CPA (10 μmol/l) after the third measurement, four of the seven sets of slices were washed out with Ca2+-free Tyrode solution for 10 min and the V˙o 2 of the same slices was measured for 6 min. These values were used as back control values.
Six sets of slices from six rats were inprotocol 4 and treated with 2,3-butanedione monoxime (BDM; 5 mmol/l). The control value was obtained as in protocol 1. After washout of the slices with Ba2+ (1 mmol/l) Tyrode solution for 10 min, the second V˙o 2measurement of Ba2+-activated slices was performed for 6 min. These values were used as Ba2+ (1 mmol/l)V˙o 2 values. After washout of the slices with Ba2+ (1 mmol/l) Tyrode solution containing BDM (5 mmol/l) for 10 min, the thirdV˙o 2 measurement in this solution was performed for 6 min. ThisV˙o 2 value was used as the BDM V˙o 2 value.
All data are presented as means ± SD. Data were analyzed by one-way and repeated-measures ANOVA and followed, as necessary, by multiple comparisons using Bonferroni t-tests. In all statistical tests, P values <0.05 were considered statistically significant.
V˙o2 for Ca2+-Free Quiescent Slices and Ba2+-Activated Slices
The V˙o 2 in Ca2+-free Tyrode solution was 2.32 ± 0.53 ml O2 ⋅ min−1 ⋅ 100 g LV−1 (basal metabolism) and was expressed as 100%. TheV˙o 2 during Ba2+ (1 mmol/l;n = 6) contracture significantly increased to 140% of basal metabolism (P < 0.05). TheV˙o 2 of Ba2+-activated slices minus the basal metabolic V˙o 2(ΔV˙o 2) was 42.5 ± 12.7% of basal metabolism. After washout of Ba2+,V˙o 2 of the same slices in Ca2+-free Tyrode solution returned to the basal metabolic V˙o 2. The present basal metabolicV˙o 2 corresponded to our previously measured constant basal metabolism in Ca2+-free or normal Tyrode solution without stimulation (29, 36). The observed full recovery from Ba2+-induced increase inV˙o 2 of myocardial slices was in accordance with the results obtained in the superfused papillary muscle and Langendorff-perfused heart; the Ba2+ contracture was attenuated as the Ba2+ was washed out of the cardiac cells when Ba2+ Tyrode solution was displaced by normal Tyrode solution (17, 23).
Concentration Dependence of Ba2+Activation of Slice V˙o2
The pooled control V˙o 2 values of quiescent slices and back control of the same slices in Ca2+-free Tyrode solution were almost constant at 2.25 ± 0.54 ml O2 ⋅ min−1 ⋅ 100 g LV−1 in all series ofprotocol 1. As shown in Fig.1, theV˙o 2 values of the slices activated by Ba2+ increased in a dose-dependent manner from 110% of control at 0.3 mmol/l Ba2+(n = 6) to 150% of control at 3 mmol/l Ba2+(n = 9). TheV˙o 2 significantly increased from that at 0.5 mmol/l Ba2+(P < 0.05) and gradually increased up to that at 3 mmol/l Ba2+. However, the V˙o 2 at 3 mmol/l Ba2+ did not significantly increase from that at 1 mmol/l Ba2+(P > 0.05). Therefore, we used 1 mmol/l Ba2+ inprotocols 2–4.
Constancy of Repeated Measurement ofV˙o2 Value in Ba2+-Activated Slices
In protocol 2, after theV˙o 2 of the quiescent slices was measured in Ca2+-free Tyrode solution (2.55 ± 0.55 ml O2 ⋅ min−1 ⋅ 100 g LV−1), we repeatedly measured the V˙o 2 of the same Ba2+-activated slices. The respective second and thirdV˙o 2 values in 1 mmol/l Ba2+ were almost constant (3.34 ± 0.57 vs. 3.41 ± 0.47 ml O2 ⋅ min−1 ⋅ 100 g LV−1). The constancy of repeatedly measured V˙o 2 during Ba2+ contracture enabled us to compare the second V˙o 2 with the third V˙o 2 following treatment with CPA (protocol 3) or BDM (protocol 4).
Effects of CPA on V˙o2 of Ba2+-Activated Slices
The control V˙o 2 in Ca2+-free Tyrode solution was 2.30 ± 0.61 ml O2 ⋅ min−1 ⋅ 100 g LV−1. TheV˙o 2 of Ba2+-activated slices significantly increased to 141.5 ± 12.1% of the controlV˙o 2(P < 0.05) (Fig.2). TheV˙o 2 of Ba2+-activated slices treated with CPA was 142.1 ± 14.1% of the controlV˙o 2 (Fig. 2). Thus CPA did not decrease the V˙o 2 of Ba2+-activated slices (P > 0.05). To confirm the recovery of Ba2+-activated slices treated with CPA, the slices were washed out with Ca2+-free Tyrode solution and the fourth V˙o 2 of the same slices in the absence of extracellular Ba2+ was measured (n = 4). The fourthV˙o 2 of the slices was 2.13 ± 0.60 ml O2 ⋅ min−1 ⋅ 100 g LV−1(n = 4). The firstV˙o 2 of basal metabolism was 1.95 ± 0.59 ml O2 ⋅ min−1 ⋅ 100 g LV−1. These values were not significantly different from each other (P > 0.05).
Effects of BDM on V˙o2 of Ba2+-Activated Slices
The V˙o 2 of Ba2+-activated slices was significantly (P < 0.05) reduced from 141.8 ± 21.3 to 110.1 ± 6.7% of the controlV˙o 2 value (2.23 ± 0.56 ml O2 ⋅ min−1 ⋅ 100 g LV−1) by treatment with BDM (Fig. 3).
The most important results are 1) our method for measurement of myocardialV˙o 2 using mechanically unloaded myocardial slices from rat LV (29, 36) showed a stable and reversible increase in V˙o 2 due to Ba2+ contracture similar to that in Langendorff-perfused hearts and superfused papillary muscle (17, 23, 24); 2) the increase inV˙o 2 by Ba2+ was dose dependent between 0.3 and 3 mmol/l; 3) the increasedV˙o 2 was not reduced by the SR Ca2+ pump inhibitor CPA (10 μmol/l); and 4) the increasedV˙o 2 was, however, reduced by the cross-bridge cycling inhibitor BDM (5 mmol/l). Therefore, an increase in V˙o 2 by Ba2+ contracture was due to increased cross-bridge cycling but not due to increased Ca2+ handling in E-C coupling mainly used for the SR Ca2+ pump (29).
A New Approach of LV Mechanoenergetics Using Mechanically Unloaded Myocardial Slices
We and others have already reported a linear myocardialV˙o 2 systolic pressure-volume area (a total mechanical energy) from the curved end-systolic pressure-volume relation in the rat LV in the blood-perfused whole heart preparation (10, 11, 31, 33), similar to that in canine and rabbit hearts (8, 18, 27, 28, 30). TheV˙o 2 intercept of this relation (minimally loaded V˙o 2, not mechanically unloaded V˙o 2) is mainly composed of the E-C couplingV˙o 2 and basal metabolicV˙o 2 (10, 11, 31). However, theV˙o 2-intercept may include theV˙o 2 residual cross-bridge cycling (12, 34). Therefore, we have proposed a new approach for evaluating the mechanically unloadedV˙o 2 using myocardial slices.
Recently, we have reported that BDM (5 mmol/l) markedly attenuated the 1-Hz twitch-free shortening of mechanically unloaded myocardial slices by 78% of control but did not decrease E-C couplingV˙o 2 (29, 34, 36). This indicates that the V˙o 2 for the residual cross-bridge cycling during mechanically unloaded twitches was negligibly small (29, 36). However, when cross-bridge cycling is enhanced, the V˙o 2 for its cycling could be detected. Actually, we obtained the expected results in the present study; the V˙o 2during Ba2+ contracture of myocardial slices increased with Ba2+ concentration in a dose-dependent manner. This increase is due to the increase inV˙o 2 for cross-bridge cycling, because E-C coupling V˙o 2 is almost completely abolished in Ca2+-free Tyrode solution (36). From these results, we conclude that the energy expenditure of myocardial slices during 1-Hz twitch-free shortening is composed of the E-C coupling and basal metabolicV˙o 2 but is not composed of the detectable V˙o 2 for the cross-bridge cycling.
Basal metabolism after KCl arrest is ∼20 μl O2 ⋅ min−1 ⋅ g−1(0.02 ml O2 ⋅ min−1 ⋅ g−1) in an intact rat heart (11). On the other hand, basal metabolicV˙o 2 (in Ca2+-free medium) of rat myocardial slices (mechanically unloaded) was ∼2.3 ml O2 ⋅ min−1 ⋅ 100 g−1 (0.023 ml O2 ⋅ min−1 ⋅ g−1) in the present study. Therefore, the basal metabolicV˙o 2 in rat myocardial slices (29, 36) is nearly equal to that in an intact heart (11). This value is greater than that in dogs and rabbits (11, 18, 29, 36). We have previously reported a lack of suppression of basal metabolism by SR Ca2+-ATPase inhibitors (29). From these results, we have already suggested energy-consuming (wasting) processes, such as protein synthesis, other than those at the SR as the possible underlying mechanism for the greater basal metabolism in the rat myocardium (29).
V˙o2 of Ba2+-Activated Slices
The present results revealed for the first time that Ba2+ increased theV˙o 2 of myocardial slices in a dose-dependent manner. These findings are reasonable considering the mechanism of Ba2+ contracture; Ba2+ interacts with troponin C binding site like Ca2+, in a dose-dependent manner, and allows cross-bridge formation (21-24), i.e., an increase in energy expenditure due to an enhanced number of cross-bridge cycles of attachment and detachment (35).
In the present experiment, theV˙o 2 began to increase by 10% (0.2 ml O2 ⋅ min−1 ⋅ 100 g LV−1) of the controlV˙o 2 of quiescent slices at 0.3 mmol/l of Ba2+, and it gradually increased up to that at 3 mmol/l Ba2+. The Ba2+ contracture of the slice was also confirmed by the microscopic observation in the present experiment. Although the steady isometric tension increased as Ba2+ increased from ∼1 μmol/l to 0.1 mmol/l in the glycerinated skinned papillary muscle (22), the effective Ba2+ concentration on LV pressure in Ba2+-perfused rabbit hearts (17) is similar to that onV˙o 2 due to Ba2+ contracture of myocardial slices. Furthermore, the muscle tension developed by Ba2+ of rabbit papillary muscle bathed in Tyrode solution reached the maximum above 4 mmol/l Ba2+ (24).
The V˙o 2 of 1 mmol/l Ba2+-activated slices pooled from all experiments corresponds to ∼140% of the controlV˙o 2 in Ca2+-free Tyrode solution. However, in the rabbit LV preloaded at its end-diastolic pressure of 10 mmHg, mean LV V˙o 2 during the first 30 min of Ba2+ contracture increased to 3.63 ml O2 ⋅ min−1 ⋅ 100 g LV−1 (∼300% of basal metabolic V˙o 2 of rabbit cardiac muscle) (24). Furthermore, Munch et al. (17) have reported a linear increase in LV pressure due to the increase in LV volume during Ba2+ contracture of rabbit hearts. These results suggest that an increase inV˙o 2 under a preloaded condition would be higher than that under a mechanically unloaded condition even in the myocardium during Ba2+ contracture.
Reversibility and Stability ofV˙o2 of Slice During Ba2+ Contracture
In the present study, basal metabolicV˙o 2 of myocardial slices under Ca2+-free conditions corresponded to that under normal Tyrode conditions without stimulation (7, 29, 36) and that under KCl arrest in the rat heart (11). TheV˙o 2 after washout of 1 mmol/l Ba2+ with Ca2+-free Tyrode solution returned to the pre-Ba2+ level ofV˙o 2 obtained by the first measurement in Ca2+-free Tyrode solution. Thus the Ba2+ that enters the cell (16) can be extruded from the myocardium (3,4).
From the measurements of ATP and phosphocreatine during Ba2+ contracture, Shibata et al. (24) supported the notion that Ba2+ induces a form of contracture that is quite stable but associated with a minimal energy demand, perhaps primarily for the cross-bridge cycling. In fact, theV˙o 2 of Ba2+ contracture of mechanically unloaded slices continued for at least 32 min in the present experiment, and the value was constant, corresponding to 130–140% of basal metabolic V˙o 2.
Effects of CPA and BDM
Saeki et al. (22, 23) claimed that there is no direct evidence that Ba2+ exclusively activates the contractile system; in fact, they observed that Ba2+ depolarized the membrane potential independently of activating the contractile system. We have already reported that CPA inhibits theV˙o 2 of the myocardial slices during 1-Hz mechanically unloaded contraction in a dose-dependent manner (1–10 μmol/l); at 10 μmol/l CPA the E-C couplingV˙o 2 was reduced by 70% (2,29, 31). The present results showing no decreases inV˙o 2 of Ba2+-activated slices by 10 μmol/l CPA support the postulate that theV˙o 2 of Ba2+-activated slices is not primarily consumed by the SR Ca2+-ATPase. These results, in agreement with many studies on skeletal muscle SR (9, 14, 15, 19, 20,32), suggest that Ba2+ is not cycled in SR of the rat myocardium and does not induce the release of residual Ca2+ from SR of the rat myocardium.
In the present study, we used BDM (5 mmol/l) to inhibit cross-bridge cycling. We have already confirmed that this concentration of BDM did not affect either the E-C coupling or basal metabolicV˙o 2 of the myocardial slices (36). BDM (5 mmol/l) decreasedV˙o 2 during Ba2+ contracture approximately to the basal metabolic V˙o 2 level, suggesting the possibility that the increase inV˙o 2 by Ba2+ contracture exclusively derived from the increased cross-bridge cycling. Taking the CPA and BDM data together, we conclude that the increase inV˙o 2 of Ba2+-activated slices is not due to SR Ca2+-ATPase but is primarily due to cross-bridge cycling.
We greatly thank Yukishige Akagi, a representative of the Okayama Rail Road Model Shop, for generously custom-making our oximetric chamber system.
Address for reprint requests and other correspondence: H. Kohzuki, Department of Physiology II, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan (E-mail:).
This study was partly supported by Grants-in-Aid 09670053, 09307029, 09470009, 10770307, 10558136, and 10877006 for Scientific Research from the Ministry of Education, Science, Sports and Culture and a 1997–1998 Frontier Research Grant for Cardiovascular System Dynamics from the Science and Technology Agency.
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