| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
-fodrin
1 Department of Physiology II and 2 Department of Surgery III, Nara Medical University, Kashihara, Nara 634-8521; 3 Department of Legal Medicine, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033; and 4 National Cardiovascular Center, Suita, Osaka 565-8565, Japan
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The
aim of the present study was to examine the mechanisms of
Ca2+ overload-induced contractile dysfunction in rat hearts
independent of ischemia and acidosis. Experiments were
performed on 30 excised cross-circulated rat heart preparations. After
hearts were exposed to high Ca2+, there was a contractile
failure associated with a parallel downward shift of the linear
relation between myocardial O2 consumption per beat and
systolic pressure-volume area (index of a total mechanical energy per
beat) in left ventricles from all seven hearts that underwent the
protocol. This result suggested a decrease in O2 consumption for total Ca2+ handling in
excitation-contraction coupling. In the hearts that underwent the high
Ca2+ protocol and had contractile failure, we found marked
proteolysis of a cytoskeleton protein, 
-fodrin, whereas other
proteins were unaffected. A calpain inhibitor suppressed the
contractile failure by high Ca2+, the decrease in
O2 consumption for total Ca2+ handling, and
membrane -fodrin degradation. We conclude that the exposure to high
Ca2+ may induce contractile dysfunction possibly by
suppressing total Ca2+ handling in excitation-contraction
coupling and degradation of membrane -fodrin via activation of calpain.
excitation-contraction coupling; oxygen consumption
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
ACUTE FAILING CANINE HEART MODELS have been produced by different procedures, such as hypercapnic acidosis (5), postacidotic stunning (8), postischemic stunning (19), and temporary Ca2+ overloading (1). These interventions have been associated with different abnormalities in cardiac mechanoenergetics, suggesting that acidosis, ischemia, free radicals, etc. contribute to the observed contractile failure. In clinical settings, perfusion with blood cardioplegia after cardiac arrest often results in stunned myocardium even though there is no evidence of ischemia or acidosis. Ca2+ overload may contribute to the stunning of these hearts during cardioplegia. The mechanisms leading to contractile dysfunction after Ca2+ overload are not well understood. Specifically, the coupling of mechanical work and energetics of the heart after exposure to high Ca2+ in the absence of ischemia and acidosis is still poorly understood.
The aims of the present study were to induce contractile dysfunction in rat hearts using an appropriate Ca2+-overloading protocol without ischemia and acidosis and to gain insight into its underlying mechanisms from the viewpoint of left ventricular (LV) mechanoenergetics. To characterize rat LV mechanoenergetics, we evaluated systolic pressure-volume (P-V) area (PVA; total mechanical energy per beat) at a midrange LV volume (PVAmLVV) and the myocardial O2 consumption per beat (VO2) intercept (PVA-independent VO2) of the linear relation between VO2 and PVA (6, 7, 22) (see METHODS). The VO2 intercept is mainly composed of VO2 for total Ca2+ handling in the excitation-contraction (E-C) coupling and basal metabolism (6, 7, 25), as in canine hearts (18, 21). The total Ca2+-handling VO2 is regulated by transsarcolemmal Ca2+ influx via the L-type Ca2+ channel (1, 17), Ca2+-induced Ca2+ release via the ryanodine receptor (19), and Ca2+ uptake via sarcoplasmic reticulum Ca2+-ATPase (7, 23, 25), Na+/Ca2+ exchanger, and Na+-K+-ATPase (11).
Furthermore, the proteolysis of cytoskeletal proteins, such as
-fodrin and ankyrin, and myofibrillar protein, such as troponin I,
which has been reported to be degraded after postischemic
reperfusion (15, 29, 31, 32), was evaluated in the
contractile failing heart using a Ca2+-overloading
protocol. The final goal was to determine how to protect against the
type of contractile failure used in this study and to develop
strategies for a more effective blood cardioplegia.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
This investigation conforms with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996).
Surgical preparation. Experiments were performed on 30 excised cross-circulated rat heart preparations as previously published (6, 27). In each experiment, three retired breeder male crj:Wistar rats weighing 530 ± 20 g (20 wk of age), purchased from Charles River Japan (Yokohama, Japan), were anesthetized with pentobarbital sodium (50 mg/kg ip) and intubated. All rats were heparinized (1,000 units iv). 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 (6).
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 a 0.5-ml precision glass syringe with fine scales (minimum scale, 0.005 ml). The unstretched balloon volume was below ~0.25-0.30 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 0.28 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. V0 was then finally determined as the volume-axis intercept of the best-fit end-systolic pressure (ESP)-V relation (ESPVR). We obtained the best-fit ESPVR using Eq. 1 (see Data Analysis) using the least-square method (DeltaGraph, DeltaPoint; Monterey, CA) on a personal computer (6, 27). Correlation coefficients of the best-fit ESPVRs were higher than 0.99 (Table 1).
|
Oxygen consumption. Myocardial VO2 was obtained as the product of the coronary flow and coronary arteriovenous O2 content difference. Total coronary blood flow was continuously measured with an electromagnetic flowmeter (MFV 3100, Nihon Kohden) placed in the middle of the coronary venous drainage tubing from the right ventricle (RV). LV thebesian flow was negligible. The coronary arteriovenous O2 content difference was continuously measured by passing all arterial and venous cross-circulation blood through the two cuvettes of a custom-made arteriovenous O2 content difference analyzer (PWA 200S, Shoe Technica; Chiba, Japan) as previously reported in detail (6). As shown previously (6, 27), the VO2-PVA relation was linear in the rat LV. Its slope represents the O2 cost of PVA, and its VO2 intercept represents PVA-independent VO2. The RV was kept collapsed by continuous hydrostatic drainage of the coronary venous return so that the RV PVA and hence PVA-dependent VO2 were assumed to be negligible (6, 27). The RV component of PVA-independent VO2 was calculated by multiplying biventricular PVA-independent VO2 in each contractile state with the ratio of RV weight divided by the sum of RV and LV weights. The RV PVA-independent VO2 (6, 27) was subtracted from the total VO2 to yield LV VO2. The LV (including the septum) and RV were weighed for normalization of LVV. They were 1.00 ± 0.12 and 0.30 ± 0.06 g (n = 30), respectively.
Experimental protocol.
The experimental protocol is shown in Fig.
1. LV pressure,
VO2, and PVA data were obtained at four to five
different volumes (mean volume range, 0.17 ± 0.02 ml/g) from 0.08 to 0.26 ml/g (Vol-run) in each heart. LVV was increased in steps up to
an end-diastolic pressure (EDP) of 10 mmHg in the control Vol-run. The
"control Vol-run" was performed without any inotropic
interventions. After the control Vol-run, a Ca2+-induced
different inotropic run (Ca2+ Ino run) was performed at
mLVV [0.16 or 0.18 ml = water volume infused into the balloon
(0.08 or 0.1 ml) plus V0 (0.08 ml)] (n = 13). Finally, a second Vol-run at the same LVVs as used in the control
Vol-run was performed after 20-min clearance of blood with a high
Ca2+ concentration (n = 7 of 13) by the
kidney of the supporter rat. In every Vol-run, a steady state, where LV
pressure, coronary arteriovenous O2 content difference, and
coronary flow were stable, was reached 2-3 min after changing LVV.
Mean values for mLVV and V0 (normalized for 1 g) were
0.17 (±0.02) and 0.08 (±0.01) ml/g, respectively (n = 13). The infusion rate of the 1% CaCl2 solution was
increased in steps up to 20-60 ml/h. This protocol was aimed to
not only obtain blood with high Ca2+ concentration but also
to prevent arrhythmia, to observe the processes of Ca2+
overload on-line, and to obtain the O2 cost of LV
contractility as described below. The blood Ca2+
concentration reached 9-15 mmol/l. After clearance of the blood with a high Ca2+ concentration for 20 min, the levels of
Ca2+ decreased to 1.1-2.5 mmol/l, and venous
PO2 values were between 39 and 45 mmHg. The
values of arterial-venous difference in lactate throughout the
experiment were between 0.19 and 0.25 mmol/l, indicating no lactate
production. pH was maintained at ~7.45.
|
-fodrin (31). Mean values for mLVV and V0
(normalized for 1 g) were 0.17 (±0.02) and 0.08 (±0.003) ml/g,
respectively (n = 7). The blood Ca2+
concentration reached 6-12 mmol/l.
In every Vol-run, Ca2+ Ino-run or Ca2+ Ino-run
with calpain inhibitor-1, a steady state was reached 2-3 min or 6 min after changing LVV or the infusion rate of 1% CaCl2
solution. In each steady state, data were sampled at 500 Hz for 2 s, and the sampling was usually repeated three times at intervals of
0.5-1 min.
Data analysis.
We attempted to fit experimentally obtained LV P-V data using the
following equations to obtain ESPVRs and EDP-V relations (EDPVRs)
![P<SUB>es</SUB><IT>=A</IT>{1<IT>−</IT>exp[−<IT>B</IT>(V<IT>−</IT>V<SUB>0</SUB>)]}](/content/vol281/issue3/fulltext/H1286/img001.gif)
|
(1) |
![P<SUB>ed</SUB><IT>=A′</IT>{exp[<IT>B′</IT>(V<IT>−</IT>V<SUB>u</SUB>)]<IT>−</IT>1}](/content/vol281/issue3/fulltext/H1286/img002.gif)
|
(2) |
Oxygen cost of LV contractility.
During and after the Ca2+ Ino-run at a given mLVV, ESP-V
and VO2 data were sampled. We obtained the
specific best-fit curves to the observed ESP at mLVV = 0.20 ml/g
(ESP0.20) and ESP (0 mmHg) at V0 using the
ESPVR function in the control Vol-run by the least-square method and
calculated PVAmLVV on a personal computer (Fig.
2) (27).
|
O2-PVA relation of Vol-run after high Ca2+ infusion shifted downward in parallel to the control
VO2- PVA relation. All of these results
indicate that a parallel VO2-PVA relation to
the control relation is obtainable under different contractile states.
Thus thin lines including all VO2-PVA data during the Ca2+ infusion in steps at the mLVV were drawn in
parallel to the control VO2-PVA relation line
(Fig. 2B). The gradually increased
VO2 intercept values (PVA-independent
VO2 values) of the thin lines proportional to
the enhanced LV contractility by low Ca2+ and a decreased
VO2 intercept value after high Ca2+
infusion were obtained by this procedure. The latter
VO2 intercept value was nearly equal to the
obtained VO2 intercept after high Ca2+ infusion (Fig. 3B).
|
V0) at mean mLVV
was 0.09 ml/g. The O2 cost of LV contractility is shown as
the slope of the linear relation of PVA-independent VO2 and eEmax (Fig. 2C)
(27). The dimensions of this cost and the LV contractility
index were the same as those in the canine heart (20, 24).
Statistics. Analysis of covariance (ANCOVA) was applied to compare the two regression lines of LV VO2 on PVA in each heart between Vol-runs in control and after high Ca2+ infusion. Comparison of paired individual values was performed with a paired t-test. Multiple comparisons were performed by ANOVA and Bonferroni's post hoc analysis. A value of P < 0.05 was considered statistically significant. All data are expressed as means ± SD.
SDS-PAGE and Western blot analysis.
LV myocardium from each heart was frozen and stored at 80°C after
the mechanoenergetic studies. The frozen hearts were homogenized in
sucrose-Tris-EGTA buffer containing 20 µmol/l leupeptin and 0.15 µmol/l pepstatin A (29). The homogenates (25 µg
protein/lane) or membrane fractions (24 µg protein/lane) were
subjected to SDS-PAGE (using 6.5% gels for -fodrin and ankyrin and
12.5% gels for troponin I and connexin43) by the method of Laemmli
(12), followed by immunoblotting according to the method
of Towbin et al. (26) with modifications
(31). 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 a 2,000-fold diluted
antibody against anti--fodrin (Biohit, Genex), anti-chicken erythrocyte ankyrin (Transformation Research), anti-troponin I (Biogenesis), and anti-connexin43 (Chemicon) for 1 h at room
temperature. An enhanced chemiluminesence Western blotting detection
kit was used to visualize the protein. The amounts of the 150- and
145-kDa fragments of -fodrin (240 kDa) were measured with an image
analyzer (Densitography AE 6900, Atto) (30). The intensity
of the bands was expressed in arbitrary units. The significance of
differences was evaluated by ANOVA with Bonferroni's post hoc analysis
or with a nonpaired t-test. A value of P < 0.05 was considered statistically significant. All data are expressed
as means ± SD.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
ESPVR and VO2-PVA relation.
Figure 2A shows control ESPVR during Vol-run. LV pressure
increased with increases in LVV. Averaged ESP at mean mLVV
(ESP0.17) was 77.2 ± 7.1% of the maximum
ESP (n = 13). VO2
increased with increases in LV pressure and LVV and also with an
increase in PVA. EDP was slightly increased at large LVVs. The best-fit
control ESPVR was an upward convex curve (Fig. 2A). Mean
best-fit parameters A (in mmHg) and B (in
ml
1) in seven hearts are summarized in Table 1. There was
a reasonably linear relation between VO2 and
PVA in the control (Fig. 2B). The mean slope and
VO2 intercept of
VO2-PVA relations are summarized in Table 1.
Changes in ESPmLVV and
VO2-PVAmLVV relations during and
after high Ca2+ intracoronary infusion.
During Ca2+ infusion up to 15-20 ml/h,
ESP0.20 gradually increased, and hence the PVA at mLVV = 0.20 ml/g (PVA0.20) gradually increased (arrow
1 in Fig. 2A). VO2 and
PVA0.20 increased on the composite
VO2-PVA0.20 line (arrow
1 in Fig. 2B). From PVAmLVV and
PVA-independent VO2 data
(VO2 intercept) during this infusion, we
obtained a linear relation between PVA-independent
VO2 and eEmax (calculated from
PVAmLVV) (solid line in Fig. 2C). The
O2 cost of LV contractility, i.e., the slope of this
relation, indicates the PVA-independent VO2 per
unit change in LV contractility (27). The mean
O2 cost of LV contractility was calculated to be 2.31 ± 0.84 (×104) µl
O2 · beat1 · mmHg1 · ml · g2
in normal hearts (n = 7). This value agreed with our
previously reported one (27).
Changes in ESPVR and VO2-PVA relation after high Ca2+ intracoronary infusion. Twenty minutes after the stop of the high Ca2+ infusion, the ESPs at all the LVVs decreased and the ESPVR curve shifted downward (Fig. 3A). The EDPVR hardly changed and thus the PVAs decreased (Fig. 3A). No negative lusitropism was found by evaluating the logistic time constant (14), 13.4 ± 2.2 ms in control versus 13.6 ± 2.2 ms after high Ca2+ infusion at all the LVVs in four hearts. The linear VO2-PVA relation shifted downward with the depressed contractile state without any changes in its slope (Fig. 3B). Each VO2 and PVA decreased from each control at all LVVs. Results from the seven hearts are summarized in Table 1. The mean PVA at mLVV = 0.17 ml/g (PVA0.17) decreased to 64% of the control (P < 0.01). The slope of the VO2-PVA relation was not significantly different from the control, but its VO2 intercept significantly decreased (P < 0.01 or 0.05 by ANCOVA) in each of the seven hearts. The mean intercept of the VO2-PVA relation in the seven hearts also was decreased by 45% (P < 0.05 by paired t-test) (Table 1). Coronary flow at mean mLVV (0.17 ± 0.02 ml/g) seemed to be sufficient after high Ca2+ infusion (2.2 ± 0.8 vs. 4.1 ± 0.8 ml/min in control), because the mean PVA0.17 decreased to 64% of the control.
Basal metabolic VO2 measured during KCl-induced arrest was unaltered after high Ca2+ infusion (26.7 ± 15.1 µl O2 · min1 · g1,
n = 5) compared with 30.4 ± 3.8. µl
O2 · min1 · g1
in the control (n = 5).
-Fodrin proteolysis in homogenates of LV myocardium after high
Ca2+ intracoronary infusion.
After postischemic reperfusion, proteolysis of
cytoskeletal protein (-fodrin), linker protein (ankyrin), or
regulatory elements of the myofilament (troponin I) in rat hearts has
been previously reported (15, 29, 31, 32). The proteolysis
of -fodrin or ankyrin is caused by activation of a neutral protease
(calpain) due to Ca2+ overload (29, 31, 32).
However, no proteolysis of ankyrin, troponin I, or gap junction protein
(connexin43) was detected in the homogenates of the high
Ca2+-infused hearts (Fig. 4).
We next examined the proteolysis of -fodrin in the homogenate of the
heart with interventions under various Ca2+-loading
conditions including high Ca2+ infusion, because -fodrin
is localized to the sarcolemma, the intercalated disks, and Z bands
(31).
|
-fodrin (240 kDa) in an intact heart, a heart subjected to time-matched Vol-runs with no Ca2+ infusion, and a low
Ca2+-infused and a high Ca2+-infused heart
(lanes A-D, respectively) is shown in Fig.
5, top. In the zero
Ca2+-infused heart, Vol-runs without any interventions were
performed two to three times for 3-4 h, during which time neither
the ESPVR (best-fit parameters A and B as well as
PVAmLVV) nor the VO2-PVA relation
(slope and VO2 intercept) were significantly
changed (n = 3). In the low Ca2+-infused
heart, a significant parallel upward shift of the
VO2-PVA relation was found (n = 5). The 150-kDa fragment was not detected in any of these hearts.
However, in the heart after clearance of blood with high
Ca2+ concentration, a large amount of the 150-kDa fragment
was detected. In this heart, the ESPVR curve was shifted downward and
the VO2-PVA relation was significantly shifted
downward in a parallel manner.
|
-fodrin from hearts subjected to the same interventions as shown in Fig. 5, top. The amount of the 150-kDa fragment
was significantly increased in seven hearts after clearance of blood with high Ca2+ concentration.
Relations between -fodrin proteolysis in membrane fractions of
LV myocardium and VO2 intercept of LV
VO2-PVA relation after high
Ca2+ intracoronary infusion.
Of 17 hearts, including an additional 10 hearts subjected to the
Ca2+-overloading protocol, 13 hearts showed markedly
decreased VO2 intercepts of the LV
VO2-PVA relations, but the remaining 4 hearts showed unchanged VO2 intercepts. -Fodrin
proteolysis in membrane fractions of LV myocardium was investigated in
5 of 13 hearts with the decreased VO2
intercepts and 4 of 4 hearts with unchanged VO2
intercepts, because -fodrin proteolysis is likely to occur at the
sarcolemma (see results shown in Figs. 4 and 5). Figure 6 shows the summarized data of the 150- and 145-kDa fragments of -fodrin (240 kDa) from the hearts. The
amount of products in the hearts with the decreased
VO2 intercepts was significantly increased but
that in the hearts with the unchanged VO2
intercepts was not significantly increased.
|
Relations between -fodrin proteolysis of LV myocardium and
VO2 intercept of LV
VO2-PVA relation after high
Ca2+ infusion with calpain inhibitor-1.
The VO2 intercept decreased without any change
in the slope of the VO2-PVA relation, and
-fodrin proteolysis markedly occurred in the seven hearts that
received high Ca2+ infusion (hatched bars in Fig.
7, B-D). In a different
group of seven hearts, after high-Ca2+ infusion with
calpain inhibitor-1, PVA0.17 did not significantly decreased. The VO2 intercept did not decrease,
and there was no change in the slope (crosshatched bars in Fig. 7,
C and D). The results from the seven hearts are
summarized in Table 1. In all of the five tested hearts of the same
seven hearts, calpain inhibitor-1 markedly suppressed -fodrin
proteolysis (crosshatched bar in Fig. 7B). A representative
set of immunoblots of the 150- and 145-kDa products of membrane
-fodrin is shown in Fig. 7A.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
In this study, we confirmed that neither the ESPVR nor the VO2-PVA relation were significantly changed during the repeated different volume-loading runs without any interventions for 3-4 h in blood-perfused rat hearts (n = 3). After a 20-min clearance of blood with high Ca2+ concentration attained by the high Ca2+-loading protocol, LV ESP, PVA, and thus LV contractility were decreased without changes in the O2 costs of PVA and LV contractility. This result indicates that we successfully induced LV contractile dysfunction in the blood-perfused rat heart by simple intracoronary infusion of high Ca2+ without acidosis (maintained at pH 7.45) and ischemia (no lactate production).
Linear VO2-PVA relation. The parallel downward shift of the VO2-PVA relation under the depressed contractility after the 20-min clearance after the high-Ca2+ infusion was comparable with that in Ca2+-overloaded canine hearts (1) despite the different Ca2+ treatment protocol.
VO2 intercept (PVA-independent VO2). The PVA-independent VO2 corresponds to the VO2 primarily for Ca2+ handling in E-C coupling and for basal metabolism (4, 6, 7, 9, 28). Basal metabolic VO2 was unaltered after high Ca2+ infusion. Therefore, the decrease in PVA-independent VO2 after high Ca2+ infusion indicated the decrease in VO2 for Ca2+ handling in E-C coupling (1, 7).
Oxygen cost of PVA. The unchanged O2 cost of PVA in the present study indicated unchanged chemomechanical energy transduction after the Ca2+-overloading protocol. These values compared well with those previously reported in canine hearts (1, 17). This suggests that there is no change in the product of the PVA-to-ATP coupling ratio in the contractile machinery and the ATP-to-VO2 coupling ratio in the mitochondria among canine hearts as well as normal and Ca2+-overloaded rat hearts (20, 24).
Oxygen cost of LV contractility. The unchanged O2 cost of LV contractility after the high Ca2+ infusion indicates the unchanged Ca2+ handling VO2 in E-C coupling per unit change in LV contractility. This suggests that there were no changes in the sensitivity of the contractile machinery for Ca2+, the Ca2+-to-ATP ratio in total Ca2+ handling, and the ATP-to-VO2 ratio in the mitochondria (20, 24). This suggests that the amount of Ca2+ handled in E-C coupling remains proportional to LV contractility.
Possible mechanisms for contractile dysfunction induced by
Ca2+ infusion without ischemia
and acidosis.
In the present study, there was a close correlation between the levels
of proteolysis of the membrane protein -fodrin and the contractile
dysfunction associated with the decreased Ca2+-handling
VO2. Furthermore, calpain inhibitor-1
(2.5-25 µmol/l) prevented the myocardium from the contractile
dysfunction caused by the Ca2+ infusion protocol, although
not completely (Table 1). This inhibitor also abolished the decrease in
Ca2+-handling VO2 and markedly
decreased the proteolysis of the membrane protein -fodrin.
Therefore, the transient Ca2+ overload seems to have caused
proteolysis of -fodrin at the sarcolemma and the decrease in
Ca2+-handling VO2 by activation of
a Ca2+-dependent protease, calpain.
-fodrin, ankyrin, or
troponin I have been reported to degrade after postischemic reperfusion (15, 29, 31, 32). However, in the
Ca2+-overloaded hearts, no proteolysis of ankyrin, troponin
I, or connexin43 was found. Only -fodrin proteolysis was found. This result seems to indicate that the Ca2+-overload protocol
induces less cellular breakdown than ischemia-reperfusion injury (15, 29, 31, 32).
It has been proposed that -fodrin maintains the integrity of the
plasma membranes as a constituent of the membrane skeleton (2,
13). Therefore, it seems likely that the degradation of
-fodrin in membrane fractions would alter the properties of ion
channels (31). The decreased Ca2+-handling
VO2 in E-C coupling with unchanged
O2 costs of PVA and LV contractility probably reflects the
decreased total amount of Ca2+ handled, which may be due to
a suppression of the transsarcolemmal Ca2+ influx.
The possibility that disruption of cytoskeletal proteins inactivate
L-type Ca2+ channels has been reported (3,
16). We speculate that the linkage of the L-type
Ca2+ channel to the membrane -fodrin acts to tether the
channel in place, which somehow modulates the basal activity of the
channel, and a loss of the linkage may impair its regulation.
We conclude that the contractile dysfunction observed in the present
study may involve the suppression of total Ca2+ handling in
E-C coupling and membrane -fodrin degradation by activation of
calpain. The addition of calpain inhibitors in blood cardioplegia may
be a promising strategy for improving the effectiveness of blood-based cardioplegia.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Roger J. Hajjar of the Massachusetts General Hospital Cardiovascular Research Center and Harvard Medical School for critical reading and advice regarding this manuscript.
| |
FOOTNOTES |
|---|
This study was partly supported by Ministry of Education, Science, Sports and Culture of Japan Grant-in-Aid for Scientific Research 11470277 and by a Ministry of Health, Labor, and Welfare of Japan 2000 Health Sciences research grant for Human Genomics and Regenerative Medicine.
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.
Received 21 March 2001; accepted in final form 7 June 2001.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Araki, J,
Takaki M,
Namba T,
Mori M,
and
Suga H.
Ca2+-free, high-Ca2+ coronary perfusion suppresses contractility and excitation-contraction coupling energy.
Am J Physiol Heart Circ Physiol
268:
H1061-H1070,
1995
2.
Bennett, V.
Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm.
Physiol Rev
70:
1029-1065,
1990
3.
Galli, A,
and
DeFelice LJ.
Inactivation of L-type Ca2+ channels in embryonic chick ventricle cells: dependence on the cytoskeletal agents colchicine and taxol.
Biophys J
67:
2296-2304,
1994
4.
Gibbs, CL,
Papadoyannis DE,
Drake AJ,
and
Noble MIM
Oxygen consumption of the nonworking and potassium chloride-arrested dog heart.
Circ Res
47:
408-417,
1980
5.
Hata, K,
Goto Y,
Kawaguchi O,
Takasago T,
Saeki A,
Nishioka T,
and
Suga H.
Hypercapnic acidosis increases oxygen cost of contractility in the dog left ventricle.
Am J Physiol Heart Circ Physiol
266:
H730-H740,
1994
6.
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,
1998a[ISI][Medline].
7.
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:
2137-2144,
1998b[ISI][Medline].
8.
Hata, K,
Takasago T,
Saeki A,
Nishioka T,
and
Goto Y.
Stunned myocardium after rapid correction of acidosis: increased oxygen cost of contractility and the role of Na+-H+ exchange system.
Circ Res
74:
794-805,
1994
9.
Higashiyama, A,
Watkins MW,
Chen Z,
and
LeWinter MM.
Preload does not influence nonmechanical O2 consumption in isolated rabbit heart.
Am J Physiol Heart Circ Physiol
266:
H1047-H1054,
1994
10.
Iwamoto, H,
Miura T,
Okamura T,
Shirakawa K,
Iwatate M,
Kawamura S,
Tatsuno H,
Ikeda Y,
and
Matsuzaki M.
Calpain inhibitor-1 reduces infarct size and DNA fragmentation of myocardium in ischemic/reperfused rat heart.
J Cardiovasc Pharmacol
33:
580-586,
1999[ISI][Medline].
11.
Janiak, R,
Lewartowski B,
and
Langer GA.
Functional coupling between sarcoplasmic reticulum and Na/Ca exchange in single myocytes of guinea-pig and rat heart.
J Mol Cell Cardiol
28:
253-264,
1996[ISI][Medline].
12.
Laemmli, UK.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
13.
Lazarides, E,
and
Nelson WJ.
Erythrocyte and brain forms of spectrin in cerebellum: distinct membrane-cytoskeletal domains in neurons.
Science
220:
1295-1296,
1983
14.
Matsubara, H,
Takaki M,
Yasuhara S,
Araki J,
and
Suga H.
Logistic time constant of isovolumic relaxation pressure-time curve in the canine left ventricle. Better alternative to exponential time constant.
Circulation
92:
2318-2326,
1995
15.
McDonough, JL,
Arrell DK,
and
Van Eyk JE.
Troponin I degradation and covalent complex formation accompanies myocardial ischemia/reperfusion injury.
Circ Res
84:
9-20,
1999
16.
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
17.
Namba, T,
Takaki M,
Araki J,
Ishioka K,
and
Suga H.
Energetics of the negative and positive inotropism of pentobarbitone sodium in the canine left ventricle.
Cardiovasc Res
28:
557-564,
1994
18.
Nozawa, T,
Yasumura Y,
Futaki S,
Tanaka N,
and
Suga H.
No significant increase in O2 consumption of KCl-arrested dog hearts with filling and dobutamine.
Am J Physiol Heart Circ Physiol
255:
H807-H812,
1988
19.
Ohgoshi, Y,
Goto Y,
Futaki S,
Yaku H,
Kawaguchi O,
and
Suga H.
Increased oxygen cost of contractility in stunned myocardium of dog.
Circ Res
69:
975-988,
1991
20.
Suga, H.
Ventricular energetics.
Physiol Rev
70:
247-277,
1990
21.
Suga, H,
Hisano R,
Goto Y,
Yamada O,
and
Igarashi Y.
Effect of positive inotropic agents on the relation between oxygen consumption and systolic pressure-volume area in canine left ventricle.
Circ Res
53:
306-318,
1983
22.
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
23.
Takaki, M,
Kohzuki H,
Kawatani Y,
Yoshida A,
Ishidate H,
and
Suga H.
Sarcoplasmic reticulum Ca2+ pump blockade decreases O2 use of unloaded rat heart slices: thapsigargin and cyclopiazonic acid.
J Mol Cell Cardiol
30:
649-659,
1998[ISI][Medline].
24.
Takaki, M,
Matsubara H,
Araki J,
Zhao LY,
Ito H,
Yasuhara S,
Fujii W,
and
Suga H.
Mechanoenergetics of acute failing hearts characterized by oxygen costs of mechanical energy and contractility.
In: New Horizons for Failing Heart Syndrome, edited by Sasayama S.. Tokyo, Japan: Springer-Verlag, 1996, p. 133-164.
25.
Takaki, M,
Tachibana H,
Hata Y,
Sakamoto T,
and
Suga H.
Mechanoenergetics of rat left ventricles in in situ and excised blood-perfused hearts and in unloaded rat left ventricular slices.
Heart Vessels Suppl
12:
100-102,
1997.
26.
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
27.
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[ISI][Medline].
28.
Yasuhara, S,
Takaki M,
Kikuta A,
Ito H,
and
Suga H.
Myocardial O2 of mechanically unloaded contraction of rat ventricular slices measured by a new approach.
Am J Physiol Heart Circ Physiol
270:
H1063-H1070,
1996
29.
Yoshida, K,
and
Harada K.
Proteolysis of erythrocyte-type and brain-type ankyrins in rat heart after postischemic reperfusion.
J Biochem (Tokyo)
122:
279-285,
1997
30.
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.
Biochim Biophys Acta
1317:
36-44,
1996[Medline].
31.
Yoshida, K,
Inui M,
Harada K,
Saido TC,
Sorimachi Y,
Ishihara T,
Kawasima 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
32.
Yoshida, K,
Sorimachi Y,
Fujiwara M,
and
Hironaka K.
Calpain is implicated in rat myocardial injury after ischemia or reperfusion.
Jpn Circ J
59:
40-48,
1995[Medline].
This article has been cited by other articles:
|
|
 |
|
  D. Shan, R. B. Marchase, and J. C. Chatham Overexpression of TRPC3 increases apoptosis but not necrosis in response to ischemia-reperfusion in adult mouse cardiomyocytes Am J Physiol Cell Physiol, March 1, 2008; 294(3): C833 - C841. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Liu, R. B. Marchase, and J. C. Chatham Increased O-GlcNAc levels during reperfusion lead to improved functional recovery and reduced calpain proteolysis Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1391 - H1399. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. S. Supinski, X. Ji, W. Wang, and L. A. Callahan The extrinsic caspase pathway modulates endotoxin-induced diaphragm contractile dysfunction J Appl Physiol, April 1, 2007; 102(4): 1649 - 1657. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Bartoli, N. Bourg, D. Stockholm, F. Raynaud, A. Delevacque, Y. Han, P. Borel, K. Seddik, N. Armande, and I. Richard A Mouse Model for Monitoring Calpain Activity under Physiological and Pathological Conditions J. Biol. Chem., December 22, 2006; 281(51): 39672 - 39680. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. S. Supinski and L. A. Callahan Caspase activation contributes to endotoxin-induced diaphragm weakness J Appl Physiol, June 1, 2006; 100(6): 1770 - 1777. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Nakajima-Takenaka, S. Sakata, S. Kato, Y. Ohga, K.-y. Murata, S. Taniguchi, and M. Takaki Detrimental effects after dobutamine infusion on rat left ventricular function: mechanical work and energetics Exp Physiol, July 1, 2005; 90(4): 635 - 644. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zatz and A. Starling Calpains and Disease N. Engl. J. Med., June 9, 2005; 352(23): 2413 - 2423. [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]
|
|
|
|
|
|
H. Hagihara, Y. Yoshikawa, Y. Ohga, C. Takenaka, K.-y. Murata, S. Taniguchi, and M. Takaki Na+/Ca2+ exchange inhibition protects the rat heart from ischemia-reperfusion injury by blocking energy-wasting processes Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1699 - H1707. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kobayashi, Y. Yoshikawa, S. Sakata, C. Takenaka, H. Hagihara, Y. Ohga, T. Abe, S. Taniguchi, and M. Takaki Left ventricular mechanoenergetics after hyperpolarized cardioplegic arrest by nicorandil and after depolarized cardioplegic arrest by KCl Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1072 - H1080. |
) and ESP-V data
at a midrange left ventricular (LV) volume [mLVV = 0.20 ml/g
(ESP0.20)] during a Ca2+ inotropism run. The
hatched area denotes a systolic pressure-volume area (PVA) at mLVV.
ESP0.20 during the Ca2+ inotropism run under
low Ca2+ (arrow 1, 
) and high
Ca2+ infusions (arrow 2, 
;
arrow 3,
) and
after a 20-min clearance after the high Ca2+ infusion
(arrow 4, 
) are shown. B: control
linear myocardial O2 consumption per beat
(VO2)-PVA relation during different
volume-loading runs (thick solid line and 
