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Departments of 1 Physiology II2 Thoracic-Cardiovascular Surgery, Nara Medical University School of Medicine, Kashihara, Nara 634-8521, Japan 3 Department of Neurology and Neurosurgery, Kanazawa Medical University, Kahoku-gun, Ishikawa 920-0293, Japan
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Abstract |
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intercept, which is composed of
for the total Ca2+ handling in excitation–contraction coupling and basal metabolism, of the
and PVA linear relation was also significantly (P < 0.05) decreased (n
= 8). The mean slope of the linear relation was unchanged in such hearts. Post-dobutamine basal metabolism was unchanged (n
= 5 of the 8 hearts). The moderate proteolysis of a cytoskeleton protein, 
-fodrin was identified (n
= 7 of the 8 hearts with the decreased
intercept), after clearance of blood dobutamine. In agreement with our hypothesis, the detrimental effect of the post-ß-adrenergic receptor stimulation was induced by a moderate concentration of dobutamine; we found systolic dysfunction due to the impairment of Ca2+ handling in excitation–contraction coupling in the rat LV and proteolysis of a cytoskeleton protein, -fodrin.
(Received 9 March 2005;
accepted after revision 18 April 2005; first published online 22 April 2005)
Corresponding author M. Takaki: Department of Physiology II, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan. Email: mtakaki{at}naramed-u.ac.jp
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Introduction |
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We have previously reported that the continuous infusion of dobutamine into the coronary artery induces positive inotropic effects but does not induce any detrimental effects in cross-circulated, excised normal rat hearts (Ohga et al. 2002) or even in Ca2+-overloaded contractile failing rat hearts (Tabayashi et al. 2002). The post-ß-adrenergic receptor stimulation effect on left ventricular (LV) mechanical work and energetics, however, has not been previously studied using this cross-circulation model (Ohgoshi et al. 1991; Ohga et al. 2002). We hypothesized that some detrimental effects on LV function would be induced as the post-ß-adrenergic receptor stimulation effect after dobutamine infusion and the following clearance of blood dobutamine. We further hypothesized that LV systolic dysfunction would be associated with impairment of excitation–contraction coupling and would be linked to proteolysis of -fodrin (Tsuji et al. 2001).
To test this hypothesis, we investigated post-dobutamine changes in LV mechanical work and energetics in the same type of rat whole heart preparations (Ohga et al. 2002; Tabayashi et al. 2002) that underwent dobutamine infusion into the coronary artery and clearance of blood dobutamine. We analysed LV mechanical work and energetics using the framework of curvilinear end-systolic pressure–volume relations (ESPVRs) and linear myocardial oxygen consumption per beat 
–systolic pressure–volume area (PVA; a measure of total mechanical energy per beat) relations (Takaki, 2004). In agreement with the hypothesis, we found the post-dobutamine detrimental effects on Ca2+ handling in excitation–contraction coupling in the rat LV, from analyses of LV mechanical work and energetics. LV systolic dysfunction associated with impairment of Ca2+ handling in excitation–contraction coupling and moderate proteolysis of -fodrin was observed as in Ca2+-overloaded contractile failing rat hearts (Tsuji et al. 2001).
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Methods |
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Surgical preparation
Experiments were performed on 15 excised, cross-circulated rat heart preparations as previously reported (Hata et al. 1998a,b; Tsuji et al. 1999, 2001; Sakata et al. 2001; Ohga et al. 2002). For each experiment, three retired breeder male crj: Wistar rats weighing 579 ± 74 g, were anaesthetized with pentobarbital sodium (50 mg kg–1 I.P.). All rats were intubated and given heparin (1000 U I.V.). One Wistar rat was used as a blood supplier. The beating heart was excised without interruption of coronary perfusion and supported by cross circulation with the metabolic supporter rat as previously reported in detail (Hata et al. 1998a,b).
The excised heart was maintained at 37°C. A thin latex balloon (balloon membrane volume, 0.08 ml) fitted into the LV was connected to a pressure transducer (Life Kit DX-312, Nihon Kohden, Tokyo, Japan) and a 0.5-ml precision glass syringe with fine scales (minimum scale, 0.005 ml). The maximal unstretched balloon volume was below approximately 0.20–0.25 ml. Thus, LV volume (LVV) was changed and measured by adjusting the intraballoon water volume with the syringe in 0.02- to 0.05-ml steps between 0.08 and 0.23 ml (LVV-loading run). Systolic unstressed volume (V0) was determined by filling the balloon to the level where peak isovolumic pressure and hence PVA were zero. The sum of intraballoon water volume and balloon material volume (0.08 ml) was used as an initial estimate of V0. This procedure was repeated during each LVV-loading run (control vol-run, dobutamine (dob) vol-run and post-dobutamine (post-dob) vol-run). V0 was then finally determined as the volume-axis intercept of the best-fit ESPVR in each vol-run. We obtained the best-fit ESPVR and end-diastolic pressure–volume relationship (EDPVR) from five to six different pressure (P)–volume (V) data with the two different exponential functions (see Table 1) by means of the least-squares method (Delta-Graph, DeltaPoint; Monterey, CA, USA) on a personal computer (Hata et al. 1998a,b; Tsuji et al. 1999, 2001). Correlation coefficients of the best-fit ESPVRs and EDPVRs were higher than 0.95.
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, and 
of the supporter rat were maintained within their physiological ranges with supplemental oxygen and sodium bicarbonate. We used only isovolumic contractions throughout this study, because the 
–PVA relation in the rat heart is considered to be always linear and independent of the isovolumic and ejecting contractions in a given heart at a given contractile state, as in the canine heart (Suga et al. 1981; Nozawa et al. 1989).
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Myocardial O2 consumption was obtained as the product of coronary flow and coronary arteriovenous O2 content difference. The measurements of coronary flow and arteriovenous O2 content difference have been reported earlier in detail (Hata et al. 1998a). Myocardial O2 consumption per beat 
was obtained as myocardial O2 consumption divided by heart rate (i.e. pacing rate). As shown previously (Hata et al. 1998a,b; Tsuji et al. 1999, 2001), the 
–PVA relation was linear in the rat LV. Its slope represents the oxygen cost of PVA, the efficiency of chemical to mechanical energy transduction (Takaki, 2004). Its 
intercept represents PVA-independent 
. The PVA-independent 
is composed of 
for Ca2+ handling in excitation–contraction coupling and basal metabolism (Takaki, 2004). Thus, the 
at a given PVA includes PVA-dependent 
for cross-bridge cycling and PVA-independent 
for Ca2+ handling in excitation–contraction coupling and basal metabolism (Takaki, 2004).
The right ventricle (RV) was kept collapsed by continuous hydrostatic drainage of the coronary venous return, so that the RV PVA and hence PVA-dependent 
were assumed to be negligible (Hata et al. 1998a,b; Tsuji et al. 1999, 2001). The RV component of PVA-independent 
was calculated by multiplying biventricular PVA-independent 
in each contractile state by the ratio of RV weight, divided by the sum of RV and LV weights. The RV PVA-independent 
(Hata et al. 1998a,b; Tsuji et al. 1999, 2001) was subtracted from the total 
to yield LV 
. The LV, including the septum, and the RV were weighed for normalizing LVV. They were 1.05 ± 0.09 and 0.26 ± 0.03 g, respectively (n
= 15).
Lactate measurements
Blood lactate was measured with Rapid Laboratory 860 (Bayer Medical Ltd, Tokyo, Japan). The values of arteriovenous lactate difference throughout the experiment including the maximum LVV loading (i.e. the maximum O2 demand), during control and dob vol-runs were between 0.19 and 0.25 mM, indicating no lactate production.
Experimental protocol
The experimental protocol is shown in Fig. 1. LV pressure (LVP), 
and PVA data during isovolumic contractions were obtained at five to six different volumes (mean volume range, 0.12 ± 0.01 ml g–1) (vol-run) in each heart. LVV was increased in steps up to an end-diastolic pressure of 10 mmHg in control vol-run. Control vol-run was performed without any inotropic interventions (control vol-run, 23 ± 5 min). After the control vol-run, a dobutamine-induced positive inotropic run (dob ino-run; 24 ± 3 min) at midrange LVV (mLVV) (0.16 ml = water volume infused into the balloon (0.08 ml) plus V0 (0.08 ml)) was performed (n
= 8). The mean value for mLVV (normalized for 1 g) was 0.16 ± 0.01 ml g–1. The infusion rate of 66 µM dobutamine was increased in steps up to 5 ml h–1 at 5–8 min intervals (n
= 8). The dobutamine vol-run (dob vol-run) was performed during dobutamine infusion at the maximum rate for 23 ± 6 min. The results obtained by the dob ino-run up to 5 ml h–1 alone were different from those obtained by the present combination protocol of dob ino-run followed by dob vol-run. Approximately 30 min (33 ± 38 min) after stopping dobutamine infusion for clearance of blood dobutamine, post-dobutamine vol-run (post-dob vol-run; 20 ± 3 min) was performed when LV end-systolic pressure (ESP), PVA and 
data were constant.
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Basal metabolism
To measure basal metabolism, cardiac arrest was induced by infusing KCl (0.5 M; final concentration, 36.7 ± 23.3 mM) into the coronary perfusion tubing at a constant rate (5–15 ml h–1, corresponding to the coronary flow) in normal hearts (n
= 5) and post-dobutamine hearts (n
= 5 of the 8 hearts). KCl concentration was adjusted in order to abolish electrical excitation, while monitoring ventricular electrocardiograms, but not to generate any KCl-induced constriction of coronary vessels (Hata et al. 1998b; Tsuji et al. 2001). 
and PVA data were obtained by minimal volume loading to avoid volume-loading effects on 
data. Basal metabolic oxygen consumption was obtained as the product of coronary flow and coronary arteriovenous oxygen content difference.
In every vol-run and ino-run, a steady state was reached 2–3 min after changing LVV or was reached 5–8 min after changing the infusion rate of dobutamine. In each steady state, data were sampled at 500 Hz for 2 s simultaneously, and the sampling was usually repeated three times at intervals of 0.5–1 min to minimize the noise derived from the measurement system. Mean values of three repeated values were used for analysis.
Data analysis
We attempted to fit the exponential equations to an experimentally obtained series of five to six paired LV P–V data to obtain a set of ESPVR and EDPVR. Thus we determined PVA by subtracting the area under the best-fit EDPVR from the area under the best-fit ESPVR (Tsuji et al. 1999; Abe et al. 2002; Ohga et al. 2002), although the area under the best-fit EDPVR was almost zero in control and post-dob hearts. In the present study, we calculated PVA at mLVV (PVAmLVV) to assess LV mechanical work based on our studies (Hata et al. 1998a; Tsuji et al. 1999, 2001; Abe et al. 2002; Ohga et al. 2002).
Polyacrylamide gel electrophoresis and Western blot analysis
Membrane proteins from the left ventricular myocardium of each heart were isolated as previously described (Tsuji et al. 2001; Ohga et al. 2002). The frozen hearts were homogenized in the sucrose-Tris-EGTA buffer and centrifuged at 1000 g for 10 min. The supernatants were centrifuged at 100 000 g for 60 min at 4°C. The pellets produced after centrifugation at 100 000 g were cellular membrane fractions and used for immunoblotting of -fodrin (240 kDa). Membrane proteins (12 µg lane–1) were separated on SDS-polyacrylamide gels (6.5%) in a minigel apparatus (Mini-PROTEAN II, Bio-Rad), and transferred to polyvinyliodene difluoride membranes. The membranes were blocked (4% Block Ace, Dainippon Pharmaceutical Co, Osaka, Japan) and then incubated with anti--fodrin antibody (1: 1000 dilution, Biohit, Genex), which binds to 240-kDa intact -fodrin and its fragments (150 kDa and 145 kDa). The antigens were detected by the luminescence method (ECL Western blotting detection kit, Amersham) with peroxidase-linked anti-mouse IgG (1: 1000 dilution). The amounts of 240-kDa -fodrin and its 150-kDa and 145-kDa fragments were measured by scanning the film and calculating the intensity of the bands by National Institute of Health (NIH) image analysis.
Histochemical and immunohistochemical studies
LV myocardium from each heart was frozen rapidly in isopentane chilled in dry ice and stored at –80°C before the study. For immunohistochemistry, 5-µm serial myocardium sections were fixed in acetone for 10 min at 4°C, rinsed in 0.01 M phosphate-buffered saline (PBS; pH 7.2) for 15 min, and then incubated for 30 min in blocking solution containing 2% bovine serum albumin and 5% normal goat serum, as previously described (Yoshida et al. 1995; Murata & Dalakas, 1999).
All sections were incubated with mouse monoclonal antibodies against -fodrin (1: 1500 (vol vol–1), Affiniti, AA6). We also used rabbit polyclonal antibodies against the 150-kDa fragment of -fodrin (SBDP150). SBDP150 (Y1176) antibody was made at Senju Pharmaceuticals (Kobe, Japan) against the new N-terminal of SBDP150 (H2N-Gly-Met-Met-Pro-Arg) (Yoshida et al. 1995) and diluted 1: 1000 (vol–1). After a 30-min wash in PBS, the sections were incubated with biotinylated goat anti-rabbit IgG for SBDP150 (Y1176) or goat anti-mouse IgG for anti--fodrin antibody, and then with fluorescein isothiocynate avidin D.
Serial sections consecutive to those processed as described were stained with haematoxylin and eosin (HE). Normal mouse IgG and rabbit serum, diluted to the same concentration as primary mouse and rabbit antibodies, were used for negative controls. The sections were photographed with a Zeiss epifluorescence microscope with an appropriate filter system.
Statistics
Comparisons of paired and unpaired individual values were performed by paired and unpaired t test, respectively. Analysis of covariance (ANCOVA) was applied to compare the two regression lines of LV 
on PVA in each heart between vol-runs in control (control vol-run) and after dobutamine infusion (post-dob vol-run). A value of P < 0.05 was considered statistically significant. All data are expressed as mean ±
S.D.
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Results |
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–PVA relation
We have previously reported that neither ESPVR nor 
–PVA relation was significantly changed during the repeated LVV-loading runs without any interventions for 3–4 h (versus about 2 h in the present protocol) in blood-perfused rat hearts (Tsuji et al. 2001). We also confirmed no significant effects of the infusion of the same volume of saline as dobutamine on the ESPVR and the 
–PVA relation in preliminary studies. In control vol-run, ESP increased with an increase in LVV during isovolumic contraction. 
increased with increases in ESP and LVV, and thus increased with an increase in PVA. As example is shown in Fig. 2, where the best-fit ESPVR (control ESPVR) was a convex curve in each heart. The mean V0, maximum end-systolic pressure (ESPmax), ESP at mLVV (ESPmLVV), PVAmLVV, and mLVV in eight hearts during control vol-run and post-dob vol-run are summarized in Table 2. A typical of the linear relations between 
and PVA is shown in Fig. 3. The mean slope and 
intercept of 
–PVA linear relations are summarized in Fig. 4A and B.
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–PVA relation in dob vol-run also shifted upwards (dob 
–PVA relation in Fig. 3A). After stopping the dobutamine infusion, ESPmLVV, PVAmLVV and 
started, and continued, to decrease from those values for dob vol-run (Figs 2 and 3B). Approximately 30 min after stopping dobutamine infusion, when ESPmLVV, PVAmLVV and 
were constant, the curved ESPVR shifted downwards (post-dob ESPVR) in this heart (Fig. 2). The post-dob mean ESPmLVV and thus mean PVAmLVV significantly decreased (n
= 8 in Table 2). The 
–PVA relation obtained in post-dob vol-run shifted downwards (post-dob 
–PVA relation) from that in control vol-run without changes in its slope in this heart (Fig. 3B). In the other seven hearts, similar results were obtained (Table 3).
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–PVA data point of control (filled circle) 
–PVA relation shifted left and downwards to each 
–PVA data point (open circle) at the same LVV of post-dob 
–PVA relation, indicating that each of PVA, PVA-dependent 
and PVA-independent 
during post-dob vol-run decreased from that during control vol-run (Fig. 3B and Ba). This result suggests that all of the mechanical work, oxygen consumption for cross-bridge cycling (PVA-dependent 
) and oxygen consumption for total Ca2+ handling in excitation–contraction coupling, plus basal metabolism (PVA-independent 
) decreased in this heart (Fig. 3B and Ba).
Figure 4 summarizes the mean slope and mean 
intercept of the 
–PVA relation during control vol-run and post-dob vol-run. The mean slope of the 
–PVA relation was not significantly different (Fig. 4A), but the mean 
intercept significantly decreased from that of the control (Fig. 4B). The slopes and 
intercepts of the 
–PVA relation in each of the eight hearts were also compared by ANCOVA (Table 3). Although only one heart showed a significant difference in slopes of the 
–PVA relation between control vol-run and post-dob vol-run, the other seven hearts showed no significant differences. The same seven hearts showed significant decreases in the 
intercept of the 
–PVA relation of post-dob vol-runs (Table 3).
Only the dob ino-run with dobutamine infusion up to 5 ml h–1, without the following dob vol-run (see Fig. 1), did not affect both of the post-dob ESPVR and 
–PVA relation in three of another six hearts, while the remaining three hearts showed the downward shift of both relations. It seems likely that the infusion time of dobutamine at the maximum rate is insufficient to show the downward shift of both relations in the six hearts.
The mean 
intercept value per min shown in Table 4 was obtained as the product of the 
intercept value per beat and the pacing rate (Table 2). There were no significant differences in the mean 
intercept values per min between normal and control (pre-dob) hearts. However, the mean post-dob 
intercept value 
per min was significantly smaller than the normal and control (pre-dob) value. The post-dob basal metabolic O2 consumption measured during KCl-induced arrest (25.1 ± 7.8 µl O2 min–1 g–1, n
= 5) was not significantly different from the normal value (31.2 ± 6.9 µl O2 min–1 g–1, n = 5), which was obtained after two or three repeated control vol-runs without any interventions (Table 4). The 
value per min in excitation–contraction coupling shown in Table 4 was obtained as the difference between the 
intercept value per min and basal metabolic O2 consumption. The post-dob mean 
value per min used in excitation–contraction coupling was significantly smaller than the normal value. The data suggest that the significant decrease in post-dob 
intercept was due to the decrease in 
used in excitation–contraction coupling.
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-fodrin proteolysis of LV myocardium after dobutamine intracoronary infusion and clearance of blood dobutamine
After high-Ca2+ intracoronary infusion, proteolysis of -fodrin associated with a downward shift of 
intercept in rat hearts has been previously reported (Tsuji et al. 2001). In the present study, the proteolysis of -fodrin was also examined, as post-dob hearts showed the downward shifts of the 
intercept. Three representative immunoblots of post-dob hearts and two representative immunoblots of normal hearts are shown in the upper panel of Fig. 4C. The percentage of the total amount of the 145-kDa and 150-kDa fragments of -fodrin to the intact -fodrin (240 kDa) plus its fragments was significantly larger in post-dob hearts (n
= 7 of the 8 hearts with the decreased 
intercept) than that in the normal heart (n
= 4; lower panel in Fig. 4C).
To identify the site of proteolysis, we performed an immunohistochemical study using the antibody against the 150-kDa fragment of -fodrin and the antibody against -fodrin (240 kDa) in another two hearts that underwent the same protocol as shown in Fig. 1 (post-dob heart) and two normal hearts without any treatments. A representative set of the results of histological examination by HE staining and immunostaining is shown in Fig. 5. From the result of HE staining, no enlargements of cardiac myocytes were identified in a post-dob heart (Fig. 5D). Although -fodrin (240 kDa) was observed at the inner cell membrane, its amount appeared to be less in the post-dob heart than in the normal heart (Fig. 5B and E). The proteolytic product (150 kDa) of -fodrin was hardly observed in the normal heart (Fig. 5C). In contrast to this normal heart, the 150-kDa proteolytic product of -fodrin has moderately spread over the cytoplasm in this post-dob heart (Fig. 5F).
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Discussion |
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and PVA (
–PVA relation), linking to proteolysis of a cytoskeleton protein, -fodrin (Tsuji et al. 2001). Dobutamine is known to activate the ß-adrenergic receptor–adenylate cyclase–cAMP–protein kinase A (PKA) signalling pathway (Opie, 1998), resulting in phosphorylation of L-type Ca2+ channels, ryanodine receptors and the sarcoplasmic reticulum (SR) Ca2+-ATPase regulator phospholamban (Ca2+ cycling proteins in excitation–contraction coupling). This signal pathway increases the intracellular Ca2+ concentration and the amplitude of LV contractions, and accelerates the kinetics of the target protein of PKA.
We tested the hypothesis that dobutamine infused into the coronary artery induced detrimental effects on LV function of the excised cross-circulated whole heart preparation after clearance of blood dobutamine. In agreement with our hypothesis, we found LV contractile failure in the heart that underwent ß-adrenergic stimulation by a moderate concentration of dobutamine and its clearance, similar to that induced by Ca2+-overloading with high-Ca2+ and its clearance (Tsuji et al. 2001). In the heart that underwent dobutamine infusion and its clearance, we found a decrease in mechanical work (PVA) and a decrease of PVA-dependent 
due to a decrease in 
used by myosin ATPase for cross-bridge cycling.
We found a decrease of PVA-independent 
with unchanged basal metabolism. This result indicates a decrease of 
used by Ca2+ cycling proteins for total Ca2+ handling in excitation–contraction coupling (Takaki, 2004). The oxygen cost of PVA (the slope of the 
–PVA relation) was unchanged, indicating that the ratio of chemomechanical energy transduction is unchanged (Takaki, 2004). Taken together with these results, the most pronounced, post-dobutamine detrimental effect is LV systolic dysfunction due to the impairment of total Ca2+ handling in excitation–contraction coupling (Takaki, 2004). Although this systolic dysfunction lasted for at least 124 ± 32 min (99–192 min), it seems likely to be transient, i.e. stunning.
We also found another post-dobutamine detrimental effect; that is, moderate proteolysis of -fodrin. There was a close correlation between the membrane -fodrin proteolysis and the decreased 
dependent on total Ca2+ handling in excitation–contraction coupling as previously reported (Tsuji et al. 2001; Kobayashi et al. 2004; Hagihara et al. 2005; Yoshikawa et al. 2005). -Fodrin is located at the inner cell membrane in normal hearts (Yoshida et al. 1995) but the proteolytic product (150 KDa) moderately spread over the cytosol and the less membrane -fodrin remained in the hearts that underwent dobutamine infusion and its clearance. Catecholamine inhibits tubulin polymerization in neonatal rat cardiomyocytes by excessive Ca2+ influx during ß1-adrenergic receptor stimulation (Hori et al. 1994). In the present study, -fodrin proteolysis was actually caused by dobutamine due to activation of a Ca2+-dependent neutral protease, calpain (Yoshida et al. 1995; Tsuji et al. 2001; Yoshikawa et al. 2005). Therefore, it is conceivable that exposure to dobutamine might have caused a transient, slightly excessive intracellular Ca2+ concentration during ß1-adrenergic receptor stimulation.
Alternatively, the direct stimulation of calpain activity by dobutamine may partly contribute to -fodrin proteolysis, as the ß-adrenergic agonist, isoprenaline (isoproterenol), has been reported to directly stimulate calpain activity (Iizuka et al. 1991).
We should be careful to extend our novel findings in normal rat hearts to the failing heart in human subjects. Nevertheless, we believe there is a risk that left ventricular mechanical work and energetics might be suppressed after clearance of blood dobutamine, even though they are expected to be enhanced in the presence of blood dobutamine.
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, LV P–V data during dob vol-run; 
, LV P–V data during dob positive inotropic-run (ino-run) at mLVV; 
, LV P–V data during dob clearance at mLVV; 
, LV P–V data during post-dob vol-run. mLVV, midrange left ventricular volume; PVA, systolic P–V area. LVP, left ventricular pressure; LVV, left ventricular volume.
–PVA relations in the same single heart 
–PVA data in control vol-run; 
–PVA data in dob vol-run; 
, 
–PVA data in dob ino-run at mLVV; 
-PVA data at mLVV during dob clearance; 
–PVA data in post-dob vol-run. A, each 
–PVA data point in control vol-run shifted right and upwards as indicated by each arrow. PVA-dependent 
is used for cross-bridge cycling. PVA-independent 
is used for Ca2+ handling in excitation–contraction coupling and basal metabolism. B, each 
–PVA data point in control vol-run shifted left and downwards as indicated by each arrow. Ba, more detailed explanation for square labelled a in B. A representative 
–PVA data point (•) in control vol-run shifted left and downwards (as indicated by arrow) to a representative 
–PVA data point (
and PVA-independent 
decreased to post-dob values, and also control PVA decreased to the post-dob value (as indicated by dotted arrow). The decrease from control 
intercept to post-dob value corresponds to the decrease of the PVA-independent 
(thick arrow). PVAmLVV, systolic pressure–volume area at midrange LVV in control vol-run. See 
intercept (B) of 
–PVA linear relations between control and post-dob hearts (n
= 8). C, 
–PVA relations in individual left ventricles
