HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 288/4/H1699    most recent 01033.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (13) Citing Articles via Google Scholar Google Scholar Articles by Hagihara, H. Articles by Takaki, M. Search for Related Content PubMed PubMed Citation Articles by Hagihara, H. Articles by Takaki, M.

Na⁺/Ca²⁺ exchange inhibition protects the rat heart from ischemia-reperfusion injury by blocking energy-wasting processes

Departments of ¹Physiology II and ²Thoracic-Cardiovascular Surgery, Nara Medical University, Kashihara, Nara; and ³Department of Neurology and Neurosurgery, Kanazawa Medical University, Kahoku-gun, Ishikawa, Japan

Submitted 8 October 2004 ; accepted in final form 22 December 2004

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
We have recently reported that exposure of rat hearts to high Ca²⁺ produces a Ca²⁺ overload-induced contractile failure in rat hearts, which was associated with proteolysis of -fodrin. We hypothesized that contractile failure after ischemia-reperfusion (I/R) is similar to that after high Ca²⁺ infusion. To test this hypothesis, we investigated left ventricular (LV) mechanical work and energetics in the cross-circulated rat hearts, which were subjected to 15 min global ischemia and 60 min reperfusion. Sixty minutes after I/R, mean systolic pressure-volume area (PVA; a total mechanical energy per beat) at midrange LV volume (mLVV) (PVA_mLVV) was significantly decreased from 5.89 ± 1.55 to 3.83 ± 1.16 mmHg·ml·beat^–1·g^–1 (n = 6). Mean myocardial oxygen consumption per beat (VO₂) intercept of (VO₂-PVA linear relation was significantly decreased from 0.21 ± 0.05 to 0.15 ± 0.03 µl O₂·beat^–1·g^–1 without change in its slope. Initial 30-min reperfusion with a Na⁺/Ca²⁺ exchanger (NCX) inhibitor KB-R7943 (KBR; 10 µmol/l) significantly reduced the decrease in mean PVA_mLVV and VO₂ intercept (n = 6). Although VO₂ for the Ca²⁺ handling was finally decreased, it transiently but significantly increased from the control for 10–15 min after I/R. This increase in VO₂ for the Ca²⁺ handling was completely blocked by KBR, suggesting an inhibition of reverse-mode NCX by KBR. -Fodrin proteolysis, which was significantly increased after I/R, was also significantly reduced by KBR. Our study shows that the contractile failure after I/R is similar to that after high Ca²⁺ infusion, although the contribution of reverse-mode NCX to the contractile failure is different. An inhibition of reverse-mode NCX during initial reperfusion protects the heart against reperfusion injury.

calcium overload; mechanoenergetics; KB-R7943; -fodrin

During ischemia, intracellular Na⁺ is accumulated via the activation of Na⁺/H⁺ exchanger by intracellular acidosis (6, 9, 10, 32). Because of the accumulated Na, during initial I/R, Na⁺/Ca²⁺ exchanger (NCX) activity results in a net reverse-mode operation thereby contributing to Ca²⁺ overload (6, 32). During the following reperfusion phase, both sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a) for uptake of Ca²⁺ and NCX in net forward-mode operation for extrusion of Ca²⁺ could be activated to decrease cytosolic Ca²⁺ (29, 34). Energy demand for extrusion of Ca²⁺ mediated via forward-mode NCX and Na⁺-K⁺ pump coupled to NCX (1 ATP:1 Ca²⁺) is twofold of that for uptake of Ca²⁺ by SERCA2a (1 ATP:2 Ca²⁺) (29). Therefore, myocardial oxygen consumption for total Ca²⁺ handling in the E-C coupling would increase when the contribution of forward-mode NCX for extrusion of Ca²⁺ is more prominent than SERCA2a.

We hypothesized that post-I/R contractile failure is similar to that induced by high Ca²⁺ infusion without acidosis and ischemia, even though the initiating mechanism for each contractile failure is different. We focused on the potential importance of NCX in generating post-I/R contractile failure. To test this hypothesis, we investigated left ventricular (LV) mechanical work and energetics using the cross-circulated rat hearts that underwent 15-min global ischemia and 60-min reperfusion with and without the NCX inhibitor KB-R7943 (KBR, Kanebo; Tokyo, Japan) (8) for 30 min. We evaluated LV mechanical work and energetics in the rat hearts by using 1) LV end-systolic pressure (ESP) and systolic pressure-volume area (PVA; a total mechanical energy per beat) at midrange LV volume (mLVV) (ESP_mLVV and PVA_mLVV, respectively), 2) myocardial oxygen consumption per beat (VO₂) intercept and slope of VO₂-PVA relation (1, 4, 5, 35); and 3) slope of PVA-independent VO₂-equivalent maximal elastance (eE_max) relation (20, 22, 34, 35). Present results indicate that 1) post-I/R contractile failure is similar to that induced by high Ca²⁺ without acidosis and ischemia, 2) the impairment of total Ca²⁺ handling in the E-C coupling in post-I/R contractile failure is caused by Ca²⁺ overload mediated through activation of reverse-mode NCX, and 3) KBR partially protected the heart against Ca²⁺ overload via inhibiting reverse-mode NCX during initial reperfusion.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
The investigation adhered to guidelines established in the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996).

Surgical preparation. Experiments were performed on 18 excised, cross-circulated rat heart preparations, as reported previously (4, 5, 35). In each experiment, three retired breeder male crj:Wistar rats weighing 577 ± 66 g (16–25 wk of age), purchased from Charles River Japan (Yokohama, Japan), were anesthetized with pentobarbital sodium (50 mg/kg ip) and used as blood supplier and metabolic supporter rats, respectively. All rats were heparinized (1,000 units iv). The beating heart was excised from other retired rats without interruption of coronary perfusion and supported by cross circulation with the metabolic supporter rat as previously reported in detail (4). The excised heart was maintained at 37°C.

A thin latex balloon (balloon material volume, 0.08 ml) fitted into the LV was connected to a pressure transducer (Life Kit DX-312, Nihon Kohden, Tokyo, Japan) and to a 0.5-ml precision glass syringe with fine scales (minimum scale: 0.005 ml). The maximum unstretched balloon volume was below 0.20–0.25 ml. Thus LVV was changed and measured by adjusting the intraballoon water volume with the syringe in 0.05-ml steps between 0.08 and 0.23 ml.

Systolic unstressed volume (V₀) was determined by filling the balloon to the level where peak isovolumic pressure and hence PVA (see Data analysis) 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 different LVV loading runs. We obtained the best-fit ESP-volume relation (ESPVR) with Eq. 1 seen in Table 1 by means of the least-squares method (Delta-Graph, DeltaPoint; Monterey, CA) on a personal computer (1, 4, 5, 34, 35). V₀ was then finally determined as the volume-axis intercept of the best-fit ESPVR. Correlation coefficients of the best-fit ESPVRs were higher than 0.98 (Table 1).

Oxygen consumption. Myocardial oxygen consumption was obtained as the product of coronary flow (CF) and coronary arteriovenous O₂ content difference (AVO₂D) (4, 35). Total CF was continuously measured with an electromagnetic flowmeter (MFV-3100, Nihon Koden, Tokyo, Japan) placed in the middle of the coronary venous drainage tubing from the right ventricle. LV thebesian flow was negligible. The coronary AVO₂D was continuously measured by passing all the arterial and venous cross-circulation blood through the cuvettes of a custom-made AVO₂D analyzer (PWA-200S, Shoe Technica; Chiba, Japan) as previously reported in detail (4, 35). The mean concentration of hemoglobin in the perfused blood was 14.5 ± 1.4 mg/dl.

As shown previously (1, 4, 5, 35), the VO₂-PVA relationship was linear in the rat LV. Its slope represents the O₂ cost of PVA, and its VO₂ intercept represents the PVA-independent VO₂. The PVA-independent VO₂ is composed of VO₂ in E-C coupling and basal metabolism (29). The right ventricle was kept collapsed by continuous hydrostatic drainage of coronary venous return so that the right ventricular PVA and hence PVA-dependent VO₂ were assumed to be negligible (4, 35). The right ventricular PVA-independent VO₂ was subtracted from the total VO₂ to yield LV VO₂ (4, 5, 34, 35). The LV (including the septum) and the right ventricle were weighed for normalization of LVV. They were 1.09 ± 0.12 and 0.32 ± 0.04 g (n = 6) in the I/R group and 1.15 ± 0.11 and 0.31 ± 0.05 g (n = 6) in the I/R treated with KBR (KBR + I/R) group. There were no significant differences in LV and right ventricular weights among the two rat groups.

Experiment protocol. LV pressure (LVP), VO₂, and PVA data during isovolumic contractions were simultaneously obtained at six to seven different LVVs in each heart (vol-run). All hearts except for six normal hearts underwent 15-min global ischemia (37°C) and 60-min reperfusion. Cardiac arrest was found after 7.0 ± 1.3 min (6–9 min) of global ischemia. At the onset of reperfusion, we removed the blood (3 ml) that remained at the coronary circulation system during ischemia from the venous return tube. During the total 60-min reperfusion, PVA-independent VO₂ data were obtained every 5–10 min under mechanically unloaded LV conditions. In the I/R group, there was no treatment during reperfusion. In the KBR + I/R group, KBR was infused at the final concentration of 10 µmol/l immediately after reperfusion for 30 min. KBR up to 10 µmol/l effectively blocks mechanical dysfunction of isolated perfused rat hearts, which is caused by the I/R or by hypoxia-reoxygenation insult without any effects on many other ion transporters or several cardiac action potential parameters (8, 17). During the following 30-min reperfusion, KBR infusion was stopped to prevent any inotropic effects (11), although it is reported that it has no effect on mechanical function of normal rat hearts (17). We confirmed that no changes in LV functions were found 30 min after 30-min KBR infusion (60 min after reperfusion) in the blood-perfused normal rat heart. The vol-runs were performed again 60 min after reperfusion in I/R (post-I/R vol-run) and KBR + I/R groups (post-KBR + I/R vol-run) (Fig. 1).

View larger version (31K): [in this window] [in a new window]   Fig. 1. Experimental protocols. I/R, ischemia-reperfusion protocol (n = 6); KBR + I/R, I/R protocol with 10 µmol/l KB-R7943 (KBR) (n = 6); BS, blood sampling for lactate analysis (n = each 6); Ca2+, infusion with 1% CaCl2 solution at 1–15 ml/h (n = each 6); KCl, 0.5 mol/l infusion at 10 ml/h to measure basal metabolism (n = each 3); IH, sampling for immunohistochemical study; WB, sampling for Western blotting study (n = 6 each).

After each normal, post-I/R and post-KBR + I/R vol-run, a Ca²⁺-induced different inotropism run (Ca²⁺ ino-run) was performed at mLVV [0.13 or 0.16 ml = 0.05 or 0.08 ml (water volume infused into the balloon) plus 0.08 ml (V₀)] during intracoronary infusion of 1% CaCl₂ solution. The infusion rate of Ca²⁺ was increased in steps up to 1–15 ml/h (n = 6 in each group). In every vol-run and ino-run, LVP, AVO₂D, and CF were stable after the LVV and infusion rate of Ca²⁺ were changed.

To measure basal metabolic oxygen consumption, cardiac arrest was induced by infusing KCl (0.5 mol/l) into the coronary perfusion tubing at a constant rate (n = 3 each) that was adjusted to abolish electrical excitation under monitoring ventricular electrocardiograms but not to generate any KCl-induced constrictions of coronary vessels. VO₂ and PVA data were obtained by minimal volume loading to avoid volume-loading effects, if any, on VO₂ data. In each steady state, data were sampled at 500 Hz for 3 s, and sampling was usually repeated three times at intervals of 0.5–1 min.

Data analysis. We attempted to fit experimentally obtained LVP-volume data to the exponential equations to obtain ESPVRs (see Table 1) and end-diastolic pressure-volume relationship (EDPVRs) and thus determine PVA by the same method as described previously in detail (4, 5, 34, 35). Based on our previous proposal (4, 5, 13, 27, 34), we obtained control ESPVR and calculated ESP_mLVV and PVA_mLVV to assess LV mechanical work and energetics in the two groups. Mean values for mLVV and V₀ (normalized for LV 1 g) were 0.15 ± 0.02 and 0.07 ± 0.01 ml/g, respectively (n = 18).

Oxygen cost of LV contractility. We obtained the specific best-fit curves for the observed ESP_mLVV and ESP (0 mmHg) at V₀ with the ESPVR function in the control vol-run by the least squares method, and we calculated PVA_mLVV during Ca²⁺ infusion on a personal computer (22, 34, 35). The parallelism of the VO₂-PVA linear relation during Ca²⁺ infusion has been confirmed in normal rat hearts (4, 22, 26, 34) and also was confirmed in the present I/R hearts (n = 3). From this parallelism, the lines including all VO₂-PVA data obtained during Ca²⁺ infusion in steps at the mLVV were drawn in parallel to the control VO₂-PVA relation line, as described previously (4, 22, 34, 35). The gradually increased VO₂-intercept values (PVA-independent VO₂ values) of the lines proportional to the enhanced LV contractility induced by Ca²⁺ were obtained by this procedure.

Our proposed index for LV contractility, eE_max, was calculated from a triangular area equivalent to PVA_mLVV (22, 34, 35). The O₂ cost of LV contractility was the slope of the relationship between PVA-independent VO₂ and eE_max (22, 34, 35).

Polyacrylamide gel electrophoresis and immunoblotting of 150- and 145-kDa fragments of -fodrin (240 kDa). We have previously reported that in high-Ca²⁺ infusion-induced, Ca²⁺-overloaded contractile failure associated with the impairment of total Ca²⁺ handling in the E-C coupling, proteolysis of -fodrin, a cytoskeleton protein, is found without proteolysis of ankyrin, connexin43, and troponin I (34). To compare with this contractile failure, we evaluated proteolysis of -fodrin in the present I/R-induced contractile failure, because there is a close correlation between the membrane -fodrin proteolysis and the impairment of the total Ca²⁺ handling (38).

LV myocardium from each heart was frozen and stored at –80°C after the mechanoenergetic studies. The frozen hearts were homogenized in the sucrose-Tris-EGTA buffer. The P₂ fractions (membrane fractions) (20 µg protein/lane) were subjected to SDS-polyacrylamide gel electrophoresis by the method of Laemmli (12), followed by immunoblotting according to the method of Towbin et al. (33) with modifications (37). The blots were blocked with 5% skim milk in a buffer containing 150 mmol/l NaCl, 10 mmol/l Tris·HCl, pH 7.4, and 0.05% Tween-20 and incubated with 2,000-fold diluted antibody against anti--fodrin (Biohit, Genex) for 1 h at room temperature. An ECL Western blotting detection kit visualized the protein. The amounts of the 150- and 145-kDa fragments of -fodrin were measured with an image analyzer (Densitography AE 6900, Atto)(36). The intensity of bands was expressed in an arbitrary unit. The statistical difference was evaluated by ANOVA with Bonferroni's post hoc 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 muscle sections were fixed in acetone for 10 min at 4°C, rinsed in 0.01 mol/l phosphate-buffered saline (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 described previously (16).

All sections were incubated with mouse monoclonal antibodies against -fodrin [1:1,500 (vol/vol), Affiniti, AA6]. We also used rabbit polyclonal antibodies against SBDP150. SBDP150 (Y1176) antibody was made at Senju Pharmaceuticals (Kobe, Japan) against the new NH₂-terminal of SBDP150 (H₂N-Gly-Met-Met-Pro-Arg) (33) and diluted 1:1,000 (vol/vol). SBDP150 (D1185) antibody (Cell Signaling Technology) was produced with a synthetic peptide corresponding to residues surrounding Asp1185 of human -fodrin and diluted 1:100 (vol/vol). After a 30-min wash in PBS, the sections were incubated with biotinylated goat anti-rabbit IgG for m-calpain, SBDP150 (Y1176), and SBDP150 (D1185) or goat anti-mouse IgG for the other antibodies and then with fluorescein isothiocynate Avidin D.

Serial sections consecutive to those processed as described were stained with hematoxylin and eosin (HE). Normal mouse IgG and rabbit serum, diluted to the same concentration as primary mouse and rabbit antibodies, were used for negative control. The sections were photographed with a Zeiss epifluorescence microscope with an appropriate filter system. LV cardiac cell size was determined by counting computerized pixels in digital image of myocyte.

Statistics. Comparison of paired and unpaired individual values was performed by paired and unpaired t-test, respectively. An analysis of covariance (ANCOVA) was applied to compare the two regression lines of LV VO₂ on PVA in each heart between control and post-I/R vol-runs and between control and post-KBR + I/R vol-runs. Multiple comparisons were performed by one-way or repeated-measures ANOVA with post hoc Bonferroni's test or exceptionally Dunnett's test. A value of P < 0.05 was considered statistically significant. All data are expressed as means ± SD.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
LV mechanoenergetics after I/R and after KBR + I/R. Figure 2 shows a representative control and post-I/R set of ESPVRs and EDPVRs, and VO₂-PVA relations without (A and B) and with KBR (C and D). Post-I/R curvilinear ESPVR markedly shifted downward and EDPVR slightly shifted upward (Fig. 2A). Post-I/R each PVA and VO₂ value at each LVV was markedly smaller than each control value, and the post-I/R linear VO₂-PVA relation shifted downward without change in the slope (Fig. 2B). On the other hand, the post-KBR + I/R set of ESPVRs and EDPVRs hardly changed (Fig. 2C). Post-KBR + I/R, each PVA and VO₂ value at each LVV were slightly smaller than each control value, and the post-KBR + I/R VO₂-PVA linear relation only slightly shifted downward without change in the slope (Fig. 2D).

View larger version (34K): [in this window] [in a new window]   Fig. 2. Post-I/R(A and B) and post-I/R + KBR infusion (C and D) left ventricular (LV) end-systolic pressure-volume relations (ESPVRs) and end-diastolic pressure-volume relations (EDPVRs) and myocardial oxygen consumption per beat (VO2)-systolic pressure-volume area (PVA) linear relations obtained by each volume-loading run (vol-run). Solid square, control; solid circle, post-I/R and post-KBR + I/R. Data are obtained after 60–90 min reperfusion. Detailed protocol (each vol-run) shown in Fig. 1.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Comparisons of systolic PVA at midrange LVV (PVAmLVV) (A) and decrease of myocardial VO2 intercept (B) of VO2-PVA linear relation obtained by each volume-loading run (vol-run) and proteolysis of -fodrin among control (normal) (C), I/R and KBR + I/R group hearts. *P < 0.05. D: two representative sets of WB of -fodrin and its proteolytic products in normal, I/R, and KBR + I/R group hearts. Data were obtained after 60-to-90 min reperfusion. Detailed protocol (each vol-run and WB) shown in Fig. 1.

Basal metabolic VO₂ did not change after I/R and after KBR + I/R compared with that in the normal hearts with no treatment (I/R: 30.0 ± 10.4 O₂·min^–1·g^–1; KBR + I/R: 29.8 ± 4.8 O₂·min^–1·g^–1; normal hearts: 30.4 ± 4.4 µl O₂·min^–1·g^–1). Therefore, the result indicated the decrease of VO₂ intercept (PVA-independent VO₂) was due to the decrease of the VO₂ used for Ca²⁺ handling in E-C coupling.

Polyacrylamide gel electrophoresis and immunoblotting of the 150- and 145-kDa fragments of -fodrin (240 kDa). Proteolysis of cytoskeleton protein -fodrin was analyzed after I/R and after KBR + I/R. A large amount of intact 240-kDa -fodrin was detected in the normal heart group, but 150- and 145-kDa proteolytic products were hardly detected. Mean percentage of the proteolytic products of -fodrin was significantly increased after I/R (n = 6) and was significantly decreased after KBR + I/R (n = 6), although it was still significantly larger than in normal hearts (n = 4) (Fig. 3C). Two representative sets of 150- and 145-kDa products of -fodrin in normal, post-I/R, and post-KBR + I/R hearts are shown in Fig. 3D. Percent changes in the PVA_mLVV and VO₂ intercept contrasted with changes in the proteolysis of -fodrin.

Time course of changes in the ratio of PVA-independent VO₂ to control. Time courses of changes in the ratio of PVA-independent VO₂ (VO₂ intercept) to control (=1.0) between the I/R and KBR + I/R group hearts were compared during 60-min reperfusion (Fig. 4). Although there were no significant differences in the time courses of changes in the ratio of PVA-independent VO₂ (VO₂ for Ca²⁺ handling and basal metabolism) during 30-min reperfusion between I/R and KBR + I/R group hearts, each ratio at 10 and 15 min after I/R was significantly higher than the control, whereas it was unchanged after KBR + I/R. SD values at 10 and 15 min after I/R were much larger than others, indicating that variable but transient energy-consuming processes responded to I/R. During the initial reperfusion for 5 min, the ratio of PVA-independent VO₂ was unchanged (see a in Fig. 4). During this phase, reverse-mode NCX would be activated, resulting in Ca²⁺ overload (6, 24, 37). The following energy-wasting process seemed to result from activation of forward-mode NCX and Na⁺-K⁺ pump coupled to forward-mode NCX. Therefore, it is predictable that the following energy-wasting process after blockade of the initial activation of reverse-mode NCX by KBR is consequently blocked. In fact, the ratio of PVA-independent VO₂ did not increase at 10 and 15 min after reperfusion in the KBR + I/R group (Fig. 4). Although there were no significant differences in the time courses of changes in the ratio of PVA-independent VO₂ from 30 to 60 min between I/R and KBR + I/R groups, the ratio gradually decreased in the I/R group but did not decrease in the KBR + I/R group. Finally, at 60 min after reperfusion, there was a significant difference in the ratio between the I/R and I/R + KBR groups (Fig. 4). We confirmed that KBR per se did not affect LV mechanical work and energetics at 30 min after 30-min KBR infusion in the normal heart (data not shown). Therefore, it is plausible that the decrease in the ratio of PVA-independent VO₂ in the I/R group was antagonized by treatment with KBR at 60 min after reperfusion.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Time course of changes in the ratio of systolic PVA-independent myocardial VO2 (VO2 intercept) to control in I/R and KBR + I/R group hearts at mechanically unloaded state. Small solid circle, each ratio of PVA-independent VO2 value to control; large solid circle, mean value in I/R group hearts; small open circle, each ratio of PVA-independent VO2 to control; large open circle, mean value in KBR + I/R group hearts. KBR infusion was performed for 30 min. *P < 0.05 vs. control by Dunnett's method; P < 0.05 vs. KBR + I/R.

The linear relations of PVA-independent VO₂ and eE_max obtained during the Ca²⁺ infusion in normal, I/R, and KBR + I/R group hearts could be superimposed (data not shown). Mean O₂ costs of LV eE_max for Ca²⁺ (the slope of PVA-independent VO₂ and eE_max relation) showed no significant differences among the hearts in control, after I/R and after KBR + I/R (Fig. 5). This indicated that VO₂ used for Ca²⁺ handling in E-C coupling per unit changes in LV eE_max was unchanged among the three groups. Therefore, post-I/R contractile failure has an unchanged O₂ cost of LV contractility like in high-Ca²⁺-induced contractile failure (34).

View larger version (33K): [in this window] [in a new window]   Fig. 5. Comparisons of LV oxygen costs of equivalent maximal elastance [contractility index (eEmax)] in normal, I/R, and KBR + I/R group hearts obtained by each different inotropism run (ino-run). I/R and KBR + I/R data were obtained after 90- to 120-min reperfusion. NS, not significant. Detailed protocol (each ino-run) is shown in Fig. 1.

Histochemical and immunohistochemical studies. A representative set of the results of histological examination by HE staining and immunostaining is shown in Fig. 6. -Fodrin (240 kDa) was observed at the inner cell membrane (Fig. 6, D–F), and the proteolytic products (150-kDa) of -fodrin were hardly observed in the normal heart without any treatments (Fig. 6G). In contrast to the normal heart, the 150-kDa proteolytic product of -fodrin has markedly spread over the cytoplasm in I/R group (Fig. 6H). The treatment with KBR largely reduced the spread of the 150-kDa proteolytic product (Fig. 6I). From the result of HE staining, the enlargement of cardiac myocytes probably due to the swelling was identified in a heart of the I/R group (Fig. 6B). The treatment with KBR largely reduced this enlargement (Fig. 6C). The mean cross-sectional area of cardiac cells of the I/R group hearts (n = 90 cells) was significantly larger than those of normal hearts (n = 210 cells) and the KBR + I/R group hearts (n = 180 cells), indicating that the treatment with KBR significantly reduced the enlargement of cardiac myocytes in the I/R group hearts (Fig. 7).

View larger version (124K): [in this window] [in a new window]   Fig. 6. Histochemistry by hematoxylin-eosin (HE) staining (A–C) and immunohistochemical staining (IH) of LV myocardial sections in a normal (D and G), I/R (E and H), and KBR + I/R group hearts (F and I). Detailed protocol (IH) is shown in Fig. 1. D–F: immunoreactivity for 240-kDa -fodrin. G–I: immunoreactivity for 150-kDa -fodrin proteolytic products.

View larger version (11K): [in this window] [in a new window]   Fig. 7. Comparisons of cross-section areas of cardiac myocytes among normal, I/R, and KBR + I/R group hearts using the same sections for IH. Detailed IH protocol is shown in Fig. 1. *P < 0.05.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

The total Ca²⁺handling in the myocardium is regulated by transsarcolemmal Ca²⁺ influx via L-type Ca²⁺ channel, Ca²⁺-induced Ca²⁺ release via the ryanodine receptor, Ca²⁺ uptake via SERCA2a, NCX, and Na⁺-K⁺ pump coupled to NCX. Recirculation fraction of Ca²⁺ is considered to be around 80% in rat hearts from previous results, including ours (3, 14, 19, 30), indicating that SERCA2a has a key role in the total Ca²⁺ handling in cardiac myocytes.

During ischemia, intracellular Na⁺ is accumulated via activation of Na⁺/H⁺ exchange by intracellular acidosis (6, 9, 10, 32). Because of the accumulated Na⁺, NCX activity during initial reperfusion (see a in Fig. 4) results in net reverse-mode operation to contributing to Ca²⁺ overload (6, 32). During the following reperfusion phase, both SERCA2a for uptake of Ca²⁺ and NCX in net forward-mode operation for extrusion of Ca²⁺ must be activated to decrease cytosolic Ca²⁺ (29, 34). Energy demand for extrusion of Ca²⁺ mediated via forward-mode NCX and Na⁺-K⁺ pump coupled to NCX (1 ATP:1 Ca²⁺) is twofold of that for the uptake of Ca²⁺ by SERCA2a (1 ATP:2 Ca²⁺) (25, 29). Therefore, myocardial oxygen consumption for total Ca²⁺ handling in the E-C coupling would increase when the contribution of forward-mode NCX for extrusion of Ca²⁺ to decrease cytosolic Ca²⁺ is more prominent than SERCA2a (recirculation fraction of Ca²⁺ is considered to be <80%). In fact, myocardial oxygen consumption for total Ca²⁺ handling in the E-C coupling (PVA-independent VO₂) increased, although only for 10 min as shown in Fig. 4.

These Ca²⁺-extruding mechanisms fail to respond sufficiently to the increased cytosolic Ca²⁺ concentration, resulting in the transient Ca²⁺ overload and thus proteolysis of membrane -fodrin by a Ca²⁺-dependent protease, calpain, activated by the transient Ca²⁺ overload (38).

In the present study, we focused on the potential importance of NCX. The NCX inhibitor KBR actually protected the heart against Ca²⁺ overload, although partially and not completely. KBR inhibited LV systolic dysfunction associated with a decreased total Ca²⁺ handling VO₂ in E-C coupling without changes in oxygen costs of PVA and LV eE_max.

There were some reports insisting that KBR inhibited both forward- and reverse-mode NCX (2, 7, 21). However, in the present study during initial reperfusion after ischemia, only reverse-mode NCX is activated (7, 24, 37). Therefore, KBR selectively blocked reverse-mode NCX during initial reperfusion. For the following reperfusion period, KBR may block NCX in net forward-mode operation.

The effects of KBR on forward-mode NCX are still controversial (7, 8, 18, 22, 23). We observed no effects of 10 µmol/l KBR on forward-mode NCX in Langendorff-perfused isolated rat hearts (our unpublished observation), whereas the so-called forward-mode NCX inhibitor 5 µmol/l 3',4'-dichlorobenzamil actually affected forward-mode NCX in the same type of preparations (14). On the other hand, 10 µmol/l KBR induced positive inotropic effects associated with increased PVA-independent VO₂ in blood-perfused normal rat hearts (data not shown). However, PVA-independent VO₂ did not increase after reperfusion in the KBR + I/R group hearts. Furthermore, we evaluated post-KBR + I/R LV mechanical work and energetics 3090 min after 30-min KBR infusion, where KBR did not induce any effects on LV mechanical work and energetics. Therefore, even if KBR has any effects on forward-mode NCX, it could not affect post-KBR + I/R (3090 min after KBR) LV mechanical work and energetics.

Histochemical studies revealed that enlargements of cardiac myocytes probably due to swelling after I/R were reduced by treatment with KBR. This result further supported that KBR protected cardiac myocytes against the I/R injury.

In accordance with our hypothesis, 15-min global ischemia and 60-min reperfusion caused LV contractile failure similar to high Ca²⁺-induced LV contractile failure (34). For the first time to our knowledge, our results showed the real-time process for the Ca²⁺ overload via reverse-mode NCX during initial reperfusion for 5 min and the real-time energy-wasting process due to Ca²⁺ extrusion via forward-mode NCX and Na⁺-K⁺ pump coupled to NCX (29, 34) during the following reperfusion for 10 min. Furthermore, we revealed that reverse-mode NCX inhibition protected the heart from Ca²⁺ overload and blocked the resultant impairment of total Ca²⁺ handling in the excitation-contraction coupling. We conclude that NCX plays a crucial role in the pathogenesis of contractile failure after ischemic-reperfusion in the present blood-perfused rat heart preparation.

The treatment with KBR after cardiac ischemia is beneficial during the initial reperfusion, as previously reported (7). Recently, SEA0400 has been reported as a potent and highly selective inhibitor of reverse-mode NCX (15, 28, 31). However, its selectivity is still controversial (2, 21). A highly selective reverse-mode NCX inhibitor would be useful to study the role of NCX in the heart and would have the therapeutic potential.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. Takaki, Dept. of Physiology II, Nara Medical Univ., 840 Shijo-cho, Kashihara, Nara 634-8521, Japan (E-mail: mtakaki{at}naramed-u.ac.jp)

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.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 

1. Abe T, Ohga Y, Tabayashi N, Kobayashi S, Sakata S, Misawa H, Tsuji T, Kohzuki H, Suga H, Taniguchi S, and Takaki M. Left ventricular diastolic dysfunction in type 2 diabetes mellitus model rats. Am J Physiol Heart Circ Physiol 282: H138–H148, 2002.[Abstract/Free Full Text]
2. Akabas MH. Na⁺/Ca²⁺ exchange inhibitors: potential drugs to mitigate the severity of ischemic injury. Mol Pharmacol 66: 8–10, 2004.[Free Full Text]
3. Banijamali HS, Gao WD, MacIntosh BR, and ter Keurs HE. Force-interval relations of twitches and cold contractures in rat cardiac trabeculae Effect of ryanodine. Circ Res 69: 937–948, 1991.[Abstract/Free Full Text]
4. Hata Y, Sakamoto T, Hosogi S, Ohe T, Suga H, and Takaki M. Linear O₂ use-pressure-volume area relation from curved end-systolic pressure-volume relation of the blood-perfused rat left ventricle. Jpn J Physiol 48: 197–204, 1998.[CrossRef][Web of Science][Medline]
5. Hata Y, Sakamoto T, Hosogi S, Ohe T, Suga H, and Takaki M. Effects of thapsigargin and KCl on the O₂ use of the excised blood-perfused rat heart. J Mol Cell Cardiol 30: 2137–2143, 1998.[CrossRef][Web of Science][Medline]
6. Imahashi K, Kusuoka H, Hashimoto K, Yoshioka J, Yamaguchi H, and Nishimura T. Intracellular sodium accumulation during ischemia as the substrate for reperfusion injury. Circ Res 84: 1401–1406, 1999.[Abstract/Free Full Text]
7. Inserte J, Garcia-Dorado D, Ruiz-Meana M, Padilla F, Barrabes JA, Pina P, Agullo L, Piper HM, and Soler-Soler J. Effect of inhibition of Na⁺/Ca²⁺ exchanger at the time of myocardial reperfusion on hypercontracture and cell death. Cardiovasc Res 55: 739–748, 2002.[Abstract/Free Full Text]
8. Iwamoto T, Watano T, and Shigekawa M. A novel isothiourea derivative selectively inhibits the reverse mode of Na⁺/Ca²⁺ exchange in cells expressing NCX1. J Biol Chem 271: 22391–22397, 1996.[Abstract/Free Full Text]
9. Jeroudi MO, Hartley CJ, and Bolli R. Myocardial reperfusion injury: role of oxygen radicals and potential therapy with antioxidants. Am J Cardiol 73: 2B–7B, 1994.[CrossRef][Medline]
10. Karmazyn M, Gan XT, Humphreys RA, Yoshida H, and Kusumoto K. The myocardial Na⁺-H⁺ exchanger: structure, regulation, and its role in heart disease. Circ Res 85: 777–786, 1999.[Abstract/Free Full Text]
11. Kurogouchi F, Furukawa Y, Zhao D, Hirose M, Nakajima K, Tsuboi M, and Chiba S. A Na⁺/Ca²⁺ exchanger inhibitor, KB-R7943, caused negative inotropic responses and negative followed by positive chronotropic responses in isolated, blood-perfused dog heart preparations. Jpn J Pharmacol 82: 155–163, 2000.[CrossRef][Medline]
12. Laemmi UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T₄. Nature 227: 680–685, 1970.[CrossRef][Medline]
13. Lee S, Ohga Y, Tachibana H, Syuu Y, Ito H, Harada M, Suga H, and Takaki M. Effects of myosin isozyme shift on curvilinearity of the left ventricular end-systolic pressure-volume relation of in situ rat hearts. Jpn J Physiol 48: 445–455, 1998.[CrossRef][Web of Science][Medline]
14. Misawa H, Kohzuki H, Sakata S, Ohga Y, and Takaki M. Oxygen wasting for Ca²⁺ extrusion activated by partial inhibition of sarcoplasmic reticulum Ca²⁺-ATPase by cyclopiazonic acid in rat left ventricles. Jpn J Physiol 51: 99–108, 2001.[CrossRef][Web of Science][Medline]
15. Mukai M, Terada H, Sugiyama S, Satoh H, and Hayashi H. Effects of a selective inhibitor of Na⁺/Ca²⁺ exchange, KB-R7943, on reoxygenation-induced injuries in guinea pig papillary muscles. J Cardiovasc Pharmacol 35: 121–128, 2000.[CrossRef][Web of Science][Medline]
16. Murata K and Dalakas MC. Expression of the costimulatory molecule BB-1, the ligands CTLA-4 and CD28, and their mRNA in inflammatory myopathies. Am J Pathol 155: 453–460, 1999.[Abstract/Free Full Text]
17. Nakamura A, Harada K, Sugimoto H, Nakajima F, and Nishimura N. Effects of KB-R7943, a novel Na⁺/Ca²⁺ exchange inhibitor, on myocardial ischemia/reperfusion injury. Folia Pharmacol Jpn 111: 105–115, 1998.[CrossRef]
18. Nakamura M, Sunagawa M, Kosugi T, and Sperelakis N. Actin filament disruption inhibits L-type Ca²⁺ channel current in cultured vascular smooth muscle cells. Am J Physiol Cell Physiol 279: C480–C487, 2000.[Abstract/Free Full Text]
19. Negretti N, O'Neill SC, and Eisner DA. The relative contributions of different intracellular and sarcolemmal systems to relaxation in rat ventricular myocytes. Cardiovasc Res 27: 1826–1830, 1993.[Abstract/Free Full Text]
20. Ohga Y, Sakata S, Takenaka C, Abe T, Tsuji T, Taniguchi S, and Takaki M. Cardiac dysfunction in terms of left ventricular mechanical work and energetics in hypothyroid rats. Am J Physiol Heart Circ Physiol 283: H631–H641, 2002.[Abstract/Free Full Text]
21. Reuter H, Henderson SA, Han T, Matsuda T, Baba A, Ross RS, Goldhaber JI, and Philipson KD. Knockout mice for pharmacological screening Testing the specificity of Na⁺-Ca²⁺ exchange inhibitors. Circ Res 91: 90–92, 2002.[Abstract/Free Full Text]
22. Sakata S, Ohga Y, Abe T, Tabayashi N, Kobayashi S, Tsuji T, Kohzuki H, Misawa H, Taniguchi S, and Takaki M. No dependency of a new index for oxygen cost of left ventricular contractility on heart rates in the blood-perfused excised rat heart. Jpn J Physiol 51: 177–185, 2001.[CrossRef][Web of Science][Medline]
23. Satoh H, Ginsburg KS, Qing K, Terada H, Hayashi H, and Bers DM. KB-R7943 block of Ca²⁺ influx via Na⁺/Ca²⁺ exchange does not alter twitches or glycoside inotropy but prevents Ca²⁺ overload in rat ventricular myocytes. Circulation 101: 1441–1446, 2000.[Abstract/Free Full Text]
24. Schäfer C, Ladilov Y, Inserte J, Schäfer M, Haffner S, Garcia-Dorado D, and Piper HM. Role of the reverse mode of the Na/Ca exchanger in reoxygenation-induced cardiomyocyte injury. Cardiovasc Res 51: 241–250, 2001.[CrossRef][Web of Science][Medline]
25. Shimizu J, Araki J, Mizuno J, Lee S, Syuu Y, Hosogi S, Mohri S, Mikane T, Takaki M, Taylor TW, and Suga H. A new integrative method to quantify total Ca²⁺ handling and futile Ca²⁺ cycling in failing hearts. Am J Physiol Heart Circ Physiol 275: H2325–H2333, 1998.[Abstract/Free Full Text]
26. Tabayashi N, Abe T, Kobayashi S, Yoshikawa Y, Sakata S, Takenaka C, Misawa H, Taniguchi S, and Takaki M. Oxygen costs of left ventricular contractility for dobutamine and Ca²⁺ in normal rat hearts and the cost for dobutamine in Ca²⁺ overload-induced failing hearts. Jpn J Physiol 52: 163–171, 2002.[CrossRef][Web of Science][Medline]
27. Tachibana H, Takaki M, Lee S, Ito H, Yamaguchi H, and Suga H. New mechanoenergetic evaluation of left ventricular contractility in in situ rat hearts. Am J Physiol Heart Circ Physiol 272: H2671–H2678, 1997.[Abstract/Free Full Text]
28. Takahashi K, Takahashi T, Suzuki T, Onishi M, Tanaka Y, Hamano-Takahashi A, Ota T, Kameo K, Matsuda T, and Baba A. Protective effects of SEA0400, a novel and selective inhibitor of the Na⁺/Ca²⁺ exchanger, on myocardial ischemia-reperfusion injuries. Eur J Pharmacol 458: 155–162, 2003.[CrossRef][Web of Science][Medline]
29. Takaki M. Left ventricular mechanoenergetics in small animals. Jpn J Physiol 54: 175–207, 2004.[CrossRef][Web of Science][Medline]
30. Takaki M, Kohzuki H, Kawatani Y, Yoshida A, Ishidate H, and Suga H. Sarcoplasmic reticulum Ca²⁺ pump blockade decreases O₂ use of unloaded contracting rat heart slices: thapsigargin and cyclopiazonic acid. J Mol Cell Cardiol 30: 649–659, 1998.[CrossRef][Web of Science][Medline]
31. Tanaka H, Nishimaru K, Aikawa T, Hirayama W, Tanaka Y, and Shigenobu K. Effect of SEA0400, a novel inhibitor of sodium-calcium exchanger, on myocardial ionic currents. Br J Pharmacol 135: 1096–1100, 2002.[CrossRef][Web of Science][Medline]
32. Tani M and Neely JR. Role of intracellular Na⁺ in Ca²⁺ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H⁺-Na⁺ and Na⁺-Ca²⁺ exchange. Circ Res 65: 1045–1056, 1989.[Abstract/Free Full Text]
33. Towbin H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354, 1979.[Abstract/Free Full Text]
34. Tsuji T, Ohga Y, Yoshikawa Y, Sakata S, Abe T, Tabayashi N, Kobayashi S, Kohzuki H, Yoshida K, Suga H, Kitamura S, Taniguchi S, and Takaki M. Rat cardiac contractile dysfunction induced by Ca²⁺ overload: possible link to the proteolysis of -fodrin. Am J Physiol Heart Circ Physiol 281: H1286–H1294, 2001.[Abstract/Free Full Text]
35. Tsuji T, Ohga Y, Yoshikawa Y, Sakata S, Kohzuki H, Misawa H, Abe T, Tabayashi N, Kobayashi S, Kitamura S, Taniguchi S, Suga H, and Takaki M. New index for oxygen cost of contractility from curved end-systolic pressure-volume relations in cross-circulated rat hearts. Jpn J Physiol 49: 513–520, 1999.[CrossRef][Web of Science][Medline]
36. Yoshida K, Hirata T, Akita Y, Mizukami Y, Yamaguchi K, Sorimachi Y, Ishihara T, and Kawashima S. Translocation of protein kinase C-, - and isoforms in ischemic rat heart. Biochim Biophys Acta 1317: 36–44, 1996.[Medline]
37. Yoshida K, Inui M, Harada K, Saido TC, Sorimachi Y, Ishihara T, Kawashima S, and Sobue K. Reperfusion of rat heart after brief ischemia induces proteolysis of calspectin (nonerythroid spectrin or fodrin) by calpain. Circ Res 77: 603–610, 1995.[Abstract/Free Full Text]
38. Yoshikawa Y, Hagihara H, Ohga Y, Nakajima-Takenaka C, Murata K, Taniguchi S, and Takaki M. Calpain inhibitor-1 protects the rat heart from ischemic-reperfusion injury: analysis by mechanical work and energetics. Am J Physiol Heart Circ Physiol 288: H0000–H0000, 2005.

This article has been cited by other articles:

				C. Nakajima-Takenaka, G.-X. Zhang, K. Obata, K. Tohne, H. Matsuyoshi, Y. Nagai, A. Nishiyama, and M. Takaki Left ventricular function of isoproterenol-induced hypertrophied rat hearts perfused with blood: mechanical work and energetics Am J Physiol Heart Circ Physiol, November 1, 2009; 297(5): H1736 - H1743. [Abstract] [Full Text] [PDF]

				J. Dunnick, P. Blackshear, G. Kissling, M. Cunningham, J. Parker, and A. Nyska Critical Pathways in Heart Function: Bis(2-chloroethoxy)methane-Induced Heart Gene Transcript Change in F344 Rats Toxicol Pathol, June 1, 2006; 34(4): 348 - 356. [Abstract] [Full Text] [PDF]

				Y. Yoshikawa, H. Hagihara, Y. Ohga, C. Nakajima-Takenaka, K.-y. Murata, S. Taniguchi, and M. Takaki Calpain inhibitor-1 protects the rat heart from ischemia-reperfusion injury: analysis by mechanical work and energetics Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1690 - H1698. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 288/4/H1699    most recent 01033.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (13) Citing Articles via Google Scholar Google Scholar Articles by Hagihara, H. Articles by Takaki, M. Search for Related Content PubMed PubMed Citation Articles by Hagihara, H. Articles by Takaki, M.