Original Contributions |
From the Department of Physiology and Biophysics (J.E.V.E., F.P., W.L., R.J.S.), College of Medicine, University of Illinois at Chicago; the Department of Physiology (J.E.V.E.), Queen's University, Kingston, Ontario, Canada; the Department of Surgery (F.P., W.L.), College of Medicine, University of Illinois at Chicago; Sanofi Diagnostics Pasteur (C.L.), Marnes-La-Coquette, France; and the Department of Biochemistry (R.S.H.), University of Alberta, Edmonton, Canada.
Correspondence to Jennifer E. Van Eyk, PhD, Department of Physiology, Queen's University, Kingston, Ontario K7L 3W6, Canada.
| Abstract |
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-actinin and troponin I (TnI) at its
C-terminus.
-Actinin and TnI were most susceptible to
ischemia, but with 60 minutes of ischemia/reperfusion,
there was also degradation of myosin light chain-1 (MLC1) involving a
clip of residues 1 to 19. The MLC1 degradation product was detected
in the reperfusion effluent (along with troponin T, tropomyosin, and
-actinin) but not in the tissue with 60 minutes of ischemia
with no reperfusion. Moreover, with ischemia the following
proteins became associated with the myofibrils: GAPDH and proteins of
the mitochondrial ATP synthase complex. Our results provide new
evidence regarding the mechanism by which ischemia/reperfusion
causes myocardial injury and support the hypothesis that an important
element in the injury is altered activity and structure of the
myofilaments.
Key Words: protein degradation myocardial ischemia/reperfusion myofilament troponin I
-actinin
| Introduction |
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Although it is clear that accumulation of metabolites and protons during hypoxia and ischemia results in an acute change in myofilament response to Ca2+,4 there is also evidence for persistent, but reversible, changes in myofilament response to Ca2+. However, the exact nature of the myofilament changes appears complex and variable. For example, differences in maximum activity and sensitivity to Ca2+ occurring in myofilaments isolated from control hearts and from hearts previously exposed to ischemia/reperfusion have been reported.5 6 7 The molecular mechanism may involve the breakdown and/or loss of myofilament proteins as well as an increased or new association of cellular cytoplasmic proteins with the myofilaments.8 9
Examination of the hearts and myofilaments from hearts stressed by
ischemia/reperfusion indicates that both myofilament regulatory
proteins7 10 11 and structural/cytoskeletal
proteins12 13 14 are vulnerable to cleavage or
loss. With mild ischemia/reperfusion, microtubules are
substantially disrupted,13 and lesions occur
within the tissues as a result of the loss of desmin,
-actinin, and
spectrin.15 16 Immunohistochemical
analysis of control and globally ischemic human left
ventricle also demonstrated that a number of myofilament proteins,
including actin, myosin, Tm, and TnT, may be lost from the
tissue.14 In particular, TnI, a key thin-filament
regulatory protein, has been shown to be proteolytically clipped during
ischemia and
ischemia/reperfusion.7 11 However,
different laboratories have drawn varying conclusions about the extent
and significance of TnI degradation.7 9 11
The present study was undertaken to clarify the connection between
myofilament dysfunction and changes at the protein level. Alterations
were correlated to specific proteins with increasing degrees of
ischemia, and it was determined whether these changes occurred
during ischemia or on reperfusion. Using a variety of
analytical approaches to identify myofilament, mitochondrial, and
cytosolic proteins affected by ischemia with and without
reperfusion, we report results that support the hypothesis that TnI is
degraded during ischemia and stunning. We also demonstrate that
-actinin is lost from myofilaments even with mild ischemia
and that severe ischemia results in the degradation of MLC1 and
further degradation of TnI. The breakdown product of cardiac TnI is
due to proteolysis at the C-terminus, whereas proteolysis of MLC1
occurs at its NH2-terminus. These changes in
myofilament structure associated with ischemia/reperfusion
appear to be important features of the depressed ability of the
myofilaments to develop force and to their altered sensitivity to
Ca2+. The present study is the first to
simultaneously study both mild and severe ischemia
with and without reperfusion and to correlate changes in myofilament
function with changes at the protein level. Various parts of our
results have been reported in abstract
form.17 18
| Materials and Methods |
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Perfusion of Isolated Rat Hearts
Cardiac function was measured in a nonrecirculating Langendorff
perfusion apparatus. Rats (250 to 350 g; average,
301±33 g) were anesthetized with sodium pentobarbital (50
mg/kg) and injected with heparin (200 U) before the heart was excised.
The hearts were quickly excised and placed in ice-cold saline. The
aorta was cannulated, and the heart was perfused at a coronary
flow of 14 mL/min with Krebs-Ringer bicarbonate buffer equilibrated
with 95% O2/5% CO2 at
37°C. The Krebs-Ringer bicarbonate buffer consisted of 100
mmol/L sodium chloride, 4.74 mmol/L potassium chloride, 1.18
mmol/L potassium dihydrogen phosphate, 1.18 mmol/L magnesium
sulfate, 1.15 mmol/L calcium chloride, 25 mmol/L sodium
bicarbonate, 11.5 mmol/L glucose, 4.92 mmol/L pyruvate, and
5.39 mmol/L fumarate, pH 7.4. The hearts were paced at 360 bpm.
All hearts were equilibrated with Krebs-Ringer bicarbonate buffer for
15 minutes before the experimental protocols described below. Hearts
were subjected to either continuous flow for 45 minutes (control), 15
minutes of no-flow ischemia, or 60 minutes of no-flow
ischemia with or without 45 minutes of reperfusion. No-flow
ischemia was produced by wrapping the hearts in an impermeable
plastic bag and submerging them in a water bath at 37°C. The
perfusion pressure was measured from a side port in the perfusion
apparatus, using a Statham P23 pressure transducer.
Perfusion pressures were 61.5±7.5 mm Hg during the 10-minute
equilibration period and 78.8±8.0 mm Hg after 45 minutes of
perfusion. The pressure during reperfusion was 90.2±17.3 and
133.5±29.1 mm Hg after 15 minutes of ischemia and 60
minutes of ischemia, respectively.
Tissue and Effluent Sample Preparation
One-minute fractions of the effluent were collected at the end
of the equilibration period. During reperfusion and the 45 minutes of
perfusion (control), fractions were collected every minute for 10
minutes and then every 3 minutes for the remainder of the protocol. The
fractions were frozen immediately at -70°C and then
freeze-dried.
Skinned Fiber Bundle Experiments
Hearts removed from the Langendorff perfusion
apparatus were immediately placed in cold relaxing buffer
containing 0.1 mmol/L EGTA, 2 mmol/L
Mg2+, 79.2 mmol/L potassium chloride,
5.0 mmol/L MgATP2-, 12 mmol/L creatine
phosphate, and 20 mmol/L MOPS, pH 7.0 (ionic strength, 150
mmol/L), plus the protease inhibitor cocktail.
Trabeculae were quickly dissected from the heart and placed
in 50% (vol/vol) glycerol and relaxing buffer, protease
inhibitor cocktail, and 10 mmol/L butanedione
monoxime. The trabeculae were used in skinned fiber bundle
experiments within a week. The remaining left ventricle was frozen on
dry ice and stored at -70°C. The fiber bundles (
100 µm in
diameter) from each rat were glued to a force transducer at one end and
to a fixed post attached to a micromanipulator. The fibers were skinned
in relaxing buffer containing 10 IU/mL creatine kinase and 1% Triton
X-100 for 30 minutes. The fibers were transferred to relaxing buffer
containing 10 IU/mL creatine kinase, and the sarcomere lengths were set
at 2.2 µm on the basis of the laser diffraction pattern.
Isometric pCa-force relations were determined by bathing the skinned
fiber bundles sequentially in solutions (10 mmol/L EGTA, 2
mmol/L Mg2+, 79.2 mmol/L potassium chloride,
5.0 mmol/L MgATP2-, 12 mmol/L creatine
phosphate, 10 IU/mL creatine kinase, and 20 mmol/L MOPS, pH 7.0
[ionic strength, 150 mmol/L]) that contained increasing
concentrations of calcium chloride to achieve pCa values from 8.0 to
4.5. All results are presented as mean±SEM. Data were
linearized using the Hill transformation, and the force/pCa relation
was fitted to the Hill equation using nonlinear regression
analysis to derive the pCa50 and Hill
coefficient. The total protein for each skinned fiber bundle was
determined using the Lowry assay.20 At the same
fiber bundle length, we found that relative tension expressed as
force/cross-sectional area (average of controls,
50
mN/mm2) was similar to force/mg protein. This
allowed an alternative comparison and analysis of relative
tension generated by fiber bundles from different rat heart
preparations with different treatments. In the figures, tension is
normalized to the maximum tension of control preparations.
SDS-PAGE and Western Blot Analysis of Tissue and
Effluent Samples
After Langendorff perfusion, left ventricular tissue
samples were skinned in 50% (vol/vol) glycerol and relaxing buffer
containing protease inhibitor cocktail. The myofibrils from
the global ischemia model and the left ventricular
tissue were homogenized in 160 mmol/L Tris, pH 8.8,
plus the protease inhibitor cocktail. The protein content
of the homogenate was determined using the Lowry assay.
Homogenized samples were diluted 2-fold with sample buffer
consisting of 2% SDS, 5 mmol/L Tris, pH 6.5, 20% sucrose, 0.05%
bromophenol blue, and 1 mmol/L ß-mercaptoethanol. Effluent
samples used for SDS-PAGE analysis were dialyzed against 1
mmol/L hydrochloric acid and 1 mmol/L ß-mercaptoethanol with
dialysis bags having a molecular weight cutoff of 6000. The samples
were then freeze-dried and taken up into 50 µL of 160 mmol/L
Tris, pH 8.0, plus the protease inhibitor cocktail and
diluted 2-fold with sample buffer. Tissue samples (30 µg of total
protein) and effluent samples (20 µg of total protein) were loaded on
a 12.5% SDS polyacrylamide gel using a Hoeffer or Bio-Rad
minigel apparatus. Gels were either stained with Coomassie
blue or transferred to nitrocellulose (Bio-Rad) using a Hoeffer
transfer system at 200 mA for 120 minutes or a Bio-Rad minitransfer
system at 100 mA for 60 minutes. Proteins were quantified on the
stained gel by densitometric scanning using an Ultrascan XL enhanced
laser densitometer (Pharmacia LKB Biotechnology). Western blot
analysis was carried out according to the method described by
Van Eyk et al.21 The monoclonal antibodies used
were anti-TnT clone JLT-12 (Sigma Chemical Co), anti
-actinin clone
EA-53 (Sigma) or anti
-actinin clone 157 (provided by Spectral
Diagnostics), anti-MLC1 (provided by Abbott Laboratories),
which recognizes amino acid residues 70 to 75, anti-Tm clone CH1
(Sigma), antisarcomeric actin clone 5C5 (Sigma), and anti-GAPDH
(Cedarline Laboratory Ltd). Three different anti-TnI antibodies were
used in the present study: anti-TnI clone 3309, which recognizes
amino acid residues 157 to 192 (provided by Dr J. Ladenson, Washington
University, St Louis, Mo), anti-TnI clone 10F2 (Mab 10F2), which
recognizes amino acid residues 189 to 199 (see epitope map; Fig 7
), and
anti-TnI peptide (P143T) 137 to 148 (Mab E2). The production of
the anti-TnI peptide (P143T) 137 to 148 (equivalent to skeletal TnI
residues 104 to 115) monoclonal antibodies, including Mab E2, has been
described by Van Eyk et al.21 Mab E2 recognizes
intact skeletal and cardiac TnI and cardiac TnI peptides containing
amino acid residues 136 to 148 (data not shown). Epitope mapping of Mab
10F2 was carried out by 12% SDS-PAGE of intact cardiac TnI and various
TnI fragments (2 to 5 µg) followed by Western blot analysis
as outlined above. Bovine cardiac TnI was purified from the troponin
complex by HPLC22 ; recombinant rat cardiac TnI
fragments 54 to 210, 1 to 188, and 1 to 199 were provided by Dr A.
Martin (University Illinois at Chicago),23 and
the synthetic skeletal TnI peptide 96 to 142, which is equivalent to
the cardiac peptide residues 129 to 175, was prepared by solid-phase
peptide synthesis.24 25
|
Amino Acid Sequencing of Tissue and Effluent Samples
Tissue and effluent samples were prepared and electrophoresed by
12.5% SDS-PAGE as described above. The proteins were transferred onto
PVDF (Bio-Rad) using 10 mmol/L
3-(cyclohexylamino)-1-propanesulfonic acid
buffer26 at 100 mA for 55 minutes using a Bio-Rad
minitransfer system. A Hewlett-Packard G1005A protein sequencer was
used to sequence the initial amino acids of selected bands from the
PVDF membrane using standard procedures (Alberta Peptide
Institute).
HPLC Analysis of Effluent
The lyophilized effluent fractions were dissolved in 1 mL of
water for every minute of perfusion. HPLC analysis of the
effluent was performed on an analytical Zorbax C8 300SB reversed-phase
column (4.6-mm internal diameterx250 mm, Chromagraphic
Specialists Inc). The HLPC system consisted of a Hewlett-Packard series
1090 liquid chromatograph coupled to a Hewlett-Packard Vectra 486
166-MHz XM processor. The proteins were eluted using an AB solvent
system. Buffer A was composed of 0.05% aqueous trifluoroacetic acid,
and buffer B was composed of 0.05% trifluoroacetic acid in
acetonitrile. The gradient consisted of an isocratic hold (100% buffer
A) for 5 minutes followed by a 2% buffer B/min linear gradient at 1
mL/min. The proteins and protein fragments were monitored at 210 nm.
The quantity of protein present in each effluent fraction was
estimated by determining the area of the peak eluted at 23 minutes. We
have previously shown that peak area is directly related to the
quantity of the protein present.27 This
method of quantification assumes that the same protein(s) is eluted at
23 minutes in the various effluent fractions from the different
protocols.
Statistical Analyses
Statistical analysis was carried out on the data
obtained from the force/pCa measurements and the densitometric
measurements of the Coomassie bluestained SDS-PAGE. Bartlett's tests
confirmed homogeneity of variances before one-way ANOVAs to determine
differences between experimental groups. When significant F values were
obtained in a given ANOVA, means were compared by Student-Newman-Keuls
tests to isolate differences. For all analyses, a value of
P
.05 was accepted as significant. All data were
presented as mean±SEM.
| Results |
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Analysis of Protein in the Reperfusion Effluent
The data depicted in Fig 1
indicate that functional alterations,
which occur during both ischemia and
ischemia/reperfusion, influence the response of the myofilament
to both Ca2+ and Fmax. An important mechanism
likely responsible for these effects is a loss or breakdown of
myofilament proteins. We therefore determined the protein composition
and any degradation products for the tissue samples of the left
ventricle and the reperfusion effluent. The amount of protein
present in the reperfusion effluent was monitored using
reversed-phase HPLC to establish experimental conditions that
represent reversible and irreversible damage. Reversed-phase
HPLC separates proteins and peptides on the basis of hydrophobicity.
The power of this approach is that, as shown
previously,27 peak area is directly related to
the amount of protein. Thus, one can estimate the amount of protein
present in each fraction of reperfusion effluent by determining the
area of a peak, which should represent one or more proteins
present in the effluent. The appearance and subsequent
disappearance of the peak in the effluent fractions during reperfusion
provide a measure of the release kinetics as well as the relative
amounts of protein present under different experimental
conditions.
Samples of the reperfusion effluent were taken every minute for the
first 10 minutes and every 3 minutes for the remaining reperfusion
period, and Fig 2
shows
representative protein elution profiles of these
fractions for the 0-, 1-, and 10-minute samples after 60 (panel A) or
15 (panel B) minutes of ischemia. The inset shows an enlarged
scale of the 0- to 1-minute fraction. Compared with 15 minutes of
ischemia, the 60-minute ischemic episode resulted in
release of many more different proteins and protein fragments. The
results from HPLC analysis were confirmed by the 12.5%
SDS-PAGE analysis of the combined samples after 60 minutes of
ischemia (Fig 3A
).
|
|
To quantify the protein in each fraction, we integrated the peak that
is eluted at 23 minutes from the reversed-phase HPLC column in each of
the reperfusion fractions collected after ischemia (Fig 2A
and 2B
). Potentially, this peak may arise from the absorbance of several
proteins that are coeluted. The peak area from each fraction
represents the amount of protein released on reperfusion during
that time period. Fig 2C
plots the area of the peak at 23 minutes for
the fractions collected with respect to time. These data
represent the release kinetics of these proteins. Several other
peaks were quantified, and their release profiles were similar to that
observed for the peak at 23 minutes (data not shown). In all cases, the
majority of the protein was released from the heart within the initial
8 minutes of reperfusion. There was no protein present in the
perfusate collected from control hearts. There is only a small
amount of protein present in the reperfusion effluent after 15
minutes of ischemia compared with the amount of protein
present after 60 minutes of ischemia. In fact, <5% of
protein released from the heart after 60 minutes of ischemia is
released and present in the effluent after 15 minutes of
ischemia. The quantity of protein from the effluent (1 to 4
minutes) after 15 minutes of ischemia was such that even when
several rats were combined, it was insufficient for SDS-PAGE or Western
blot analysis. This suggests that there has been no breach in
membrane integrity after 15 minutes of ischemia, a hallmark for
irreversible damage. It is well established that myofilament proteins
and cytoplasmic proteins, such as creatine kinase, are lost from hearts
exhibiting myocyte necrosis, loss of membrane integrity, and
irreversible damage to the cell. This is the basis for the serum
diagnostic for myocardial infraction (for review, see
References 28 and 2928 29 ). In this experimental isolated heart model, the
release of myofilament proteins and their detection in the reperfusion
effluent would suggest that irreversible damage had occurred at 60 and
not 15 minutes of ischemia. Therefore, the change from reversible to
irreversible damage occurs after 15 minutes of ischemia.
Fig 3
illustrates 12.5% SDS-PAGE and Western blot analysis of
proteins released into the perfusate during the initial 4
minutes of reperfusion after 60 minutes of ischemia. The
myofilament proteins
-actinin, TnT, Tm, MLC1, and an MLC1
degradation product were detected in the effluent. Intact TnI was
not detected in the effluent (Fig 3E
). In addition to myofilament
proteins, the cytoplasmic proteins GAPDH (identified by Western blot,
data not shown), serum albumin, and triose phosphate isomerase
(identified by amino acid sequencing, Table 2
) were present in the effluent.
|
Analysis of Proteins in Left Ventricular
Tissue Samples
To determine whether the functional changes in skinned fiber
bundles from hearts perfused under a variety of conditions are
correlated with degradation or loss of myofilament proteins, SDS-PAGE,
Western blot analysis, and amino acid sequencing were used to
analyze the state of the myofilament proteins. Fig 4
shows representative
12.5% SDS-PAGE and Western blot analysis of left
ventricular tissue samples from hearts subjected to 45
minutes of control perfusion, 15 minutes of ischemia followed
by 45 minutes of reperfusion, 60 minutes of ischemia, or 60
minutes of ischemia followed by 45 minutes of reperfusion.
Western blots using antibodies against
-actinin, Tm, and TnT,
proteins that are present in the reperfusion effluent after 60
minutes of ischemia (Fig 3
), revealed that these proteins were
not degraded. MLC2 was also present in the tissue as an intact
protein. Thus, loss of these proteins from the cell was not the result
of degradation. Data shown in Figs 3
and 4
indicate that this does not
appear to be the case for TnI, MLC1, and
-actinin.
|
As determined by densitometric measurement of protein profiles on
12.5% gels of skinned tissue samples (Fig 5A
and 5B
, Table 1
), there was a 37%
drop in the amount of protein migrating at the same mobility as
-actinin. This loss of
-actinin occurred with 15-minute
ischemia/45-minute reperfusion, 60-minute ischemia, and
60-minute ischemia/45-minute reperfusion. No loss of protein
occurred with 15-minute ischemia alone (data not shown),
indicating that
-actinin loss from the tissue was augmented by
reperfusion. Quantification of the amount of
-actinin in the tissue
samples using Western blot analysis (Fig 4
) indicated that
densitometric analysis (Fig 5
, Table 1
) overestimated the
amount of protein. It is therefore likely that there was another
protein or fragment comigrating with
-actinin in the samples from
the ischemic/reperfused hearts. We recognize that our
conclusion is subject to potential inaccuracies in the quantification
of the protein using Western blot analysis. However,
-actinin was also lost from isolated myofibrils prepared from
globally ischemic rat hearts (Fig 6
).
|
|
Using 10% SDS-PAGE, we could detect degradation of
-actinin in the
tissue from hearts exposed to 60 minutes of ischemia (Fig 4g
).
However, with reperfusion, no degradation product could be detected
in the small amount of
-actinin left in the tissue or in the
reperfusion effluent. Previous failures to detect loss of
-actinin
by SDS-PAGE analysis during ischemia/reperfusion injury
could be due to reperfusion of the tissue, preparation of the tissue
samples (not skinned), and use of densitometry of Coomassie
bluestained gel. In our case, densitometry of Coomassie bluestained
gels detected only a small percentage of the loss of
-actinin
compared with that observed by Western blot analysis (compare
Fig 5B
or Table 1
with Fig 4
). In unskinned tissue samples, intact and
degraded
-actinin could not be differentiated using 12.5% SDS-PAGE
(data not shown). However, when the muscle cell membranes were removed
(1% Triton X-100 and/or 50% glycerol) before preparing the samples
for gel electrophoresis, the susceptibility of
-actinin to loss and
degradation became apparent by Western blot analysis (Fig 4
).
Isolated myofibrils from globally ischemic rat hearts
demonstrated the same loss of
-actinin compared with control
myofibrils (Fig 6
).
TnI degradation was observed by Western blot analysis in tissue
obtained from 60-minute ischemic and 15- and 60-minute
ischemic/reperfused hearts (Fig 4
). TnI degradation appears to
be a controlled and specific digestion that produced a single
degradation product after 15-minute ischemia/45-minute
reperfusion or 60-minute ischemia alone. However, there were at
least two degradation products after 60 minutes of ischemia
and 45 minutes of reperfusion. TnI degradation was also observed in
isolated myofibrils from globally ischemic hearts (Fig 6
). In
both cases the TnI degradation product(s) comigrated on a 12.5%
SDS-polyacrylamide gel at the same location as intact MLC1.
This affected the ability to quantify the amount of protein by SDS-PAGE
densitometry under the band that migrated at the position of intact
MLC. In fact, the amount of protein detected at that molecular weight
was greater in ischemic than in control tissue (Fig 5B
, Table 1
). This is most likely due to the combination of intact MLC (at
reduced levels as a result of degradation and loss) and the TnI
degradation product. By densitometric analysis of the
12.5% SDS-PAGE (Fig 5B
, Table 1
), both MLC1 and TnI increased with 60
minutes of ischemia followed by 45 minutes of reperfusion.
Densitometric analysis of detergent extracted muscle tissue
from hearts subjected to 15 or 60 minutes of ischemia followed
by 45 minutes of reperfusion revealed an increase in protein migrating
with the mobility of TnI (Table 1
, Fig 5C
). This reemphasizes the
difficulty in detecting and quantifying changes in the myofilament
proteins using SDS-PAGE and densitometry, which may lead to erroneous
conclusions. However, since two anti-TnI monoclonal antibodies (E2 and
3309) recognizing different epitope sequences within cardiac TnI are
able to detect the TnI degradation products, there can be no
mistake as to the identity of the degradation product.
To characterize the TnI degradation product, N-terminal amino acid
sequencing was carried out (Table 2
). Since the N-termini of both TnI
and MLC1 are acetylated, amino acid sequencing would yield the
TnI fragment only if degradation occurred within the N-terminus.
Because sequencing only detected low levels of unacetylated
MLC1, neither sequencing nor the quantity of protein available for
sequencing was limiting. This indicates that the N-terminus of TnI
fragment is acetylated and that the cleavage has occurred at
the C-terminus. Furthermore, we determined the epitope of the anti-TnI
antibody (Mab 10F2), which binds strongly to intact TnI and only weakly
to the degradation product (Fig 7A
).
Mab 10F2 epitope lies within amino acid residues 188 to 199 (Fig 7B
),
indicating that the TnI degradation product does not contain all or
part of this sequence.
Degradation of MLC1 was detected in myofibrils isolated from globally
ischemic hearts using Western blot analysis (Fig 6
).
Amino acid sequencing of this band on PVDF membrane identified the
fragment to be residues 20 to 199 (Table 2
). What is interesting is
that the degradation product was not seen in any of the
ischemic tissue samples (Fig 4
) but that it was found in the
reperfusion effluent collected after 60 minutes of ischemia
(Fig 2
). This suggests that MLC1 degradation occurs with severe
ischemia as seen in the globally ischemic hearts. The
ischemic injury to the perfused heart after 60 minutes of
ischemia appears not to be as severe, inasmuch as there was no
MLC1 degradation. However, our data indicate that reperfusion augmented
the damage already in evidence after 60 minutes of ischemia and
was sufficient to result in the degradation of MLC1.
Nonmyofilament proteins associated with the ischemic and
ischemic/reperfused tissues were also found. As previously
reported,8 9 the cytoplasmic protein, GAPDH, was
present in all ischemic tissue samples (data not shown). As
well, amino acid sequencing from the PVDF membrane of the 12.5%
SDS-PAGE of the 60-minute ischemia/45-minute reperfused tissue
sample identified two mitochondrial proteins present in the
prepared tissue sample (Table 2
). These proteins were identified as the
ATP synthase
chain and the ATP synthase OSC protein. These proteins
migrated in two bands, which became more prominent in the 60-minute
ischemia/45-minute reperfusion sample than in the control
tissue sample (Fig 5A
, Table 1
). However, the other proteins, which
have their N-terminus blocked (and thus cannot be detected by amino
acid sequencing) may also contribute to this increase in protein at
these bands. It has been reported that
/ß crystallin migrates at
the same location as the ATP synthase OSC
protein.8 9 Thus, the increase in the protein
migrating at this molecular weight could result from contributions from
both of these proteins and, possibly, from other unidentified
protein(s).
| Discussion |
|---|
|
|
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-actinin loss to
ischemia/reperfusion, (2) the effects on the myofilaments'
progress with increasing severity of ischemia/reperfusion, such
that there is MLC1 degradation and an increase in the number of TnI
degradation products, and (3) the identification of the TnI and
MLC1 degradation products. These results confirm and extend
previous data indicating that proteins not normally associated with
myofilaments are retained and apparently bound by the myofilaments even
after the skinning procedure. These changes in myofilament structure
provide a plausible mechanism for the functional changes associated
with brief ischemic episodes and for the more severe changes in
function associated with prolonged ischemia.
Functional Changes Induced in Myofilaments Stressed by
Ischemia/Reperfusion
There is a spectrum of results regarding the extent and nature of
myofilament dysfunction associated with mild and severe
ischemia/reperfusion injury. This is most likely due to
variations in the species studied, the duration of the
ischemia, and whether in situ or isolated heart models were
studied. Nevertheless, there is general agreement that myofilament
response to Ca2+ is altered. This study is the
first to examine simultaneously both mild (15-minute) and
severe (60-minute) ischemia with and without reperfusion using
the same experimental animal model. Compared with control preparations,
there was a reduction in maximum tension generated by myofilaments of
isolated rat hearts subjected to 15 minutes of ischemia with
reperfusion. These myofilaments were slightly more sensitive to
Ca2+ (Table 1
, Fig 1
). Although Gao et
al,7 30 who also studied the effects of
ischemia on rat heart preparations, reported a decrease in
maximum tension, there was a small decrease in sensitivity to
Ca2+. Skinned myocytes isolated from pig hearts
after 45 minutes of low-flow in situ ischemia with reperfusion
(stunning) also showed a decrease in Ca2+
sensitivity but no change in maximum force generation compared with the
control myocytes.6 31 This
laboratory32 has also indicated that although
stunning had little effect on the steady-state force, it may alter
crossbridge kinetics, shifting it toward force-generating states.
However, skinned myocytes by the nature of their isolation
represent a selective population of cells compared with skinned
trabecular muscle fiber bundles, which are inclusive of the
populations represented in the left ventricle at the time
of experiment; thus, these two experimental models may show differences
in their response to Ca2+. Interestingly, as was
the case in the present study using skinned trabecular
fiber bundles (Fig 1
, Table 1
), Miller et al,31
using myocytes isolated from stunned in situ hearts, showed that the
major functional damage occurs on reperfusion.
With more severe ischemia (60-minute ischemia/45-minute
reperfusion), the present study showed that the isolated skinned
trabecular fiber bundles display a relatively large
decrease in maximum force and a significant increase in
Ca2+ sensitivity compared with control fiber
bundles (Table 1
, Fig 1
). Arner et al5 also found
an increase in Ca2+ sensitivity with skinned
trabecular fiber bundles obtained from in situ global
ischemia in dog hearts. However, Dietrich et
al33 found little or no change in
Ca2+ sensitivity or maximum force in skinned
trabecular fiber bundles obtained from isolated rat hearts
that underwent 40-minute ischemia/30-minute reperfusion.
These variations in the effects of ischemia and reperfusion on myofilament function may have been due to the influence of small variations in ischemic duration that gave rise to large effects on the severity or nature of the injury that occurs. For example, Gao et al30 applied 20 minutes of no-flow ischemia, which produced a different effect than the 15 minutes of ischemia that was used in the present experiments. With 15 minutes of ischemia, there was little release of protein and no TnT release from the heart on reperfusion. However, Yamahara et al34 showed that TnT is released from isolated hearts after 20 minutes of ischemia, indicating some necrosis. Even small differences in the quantities of either intact troponin (complex of TnT, TnI, and TnC), TnI, TnC, or MLC2 lost from the myofilaments could dramatically affect myofilament response to Ca2+.35 36 37 38 39 Previous data from one of our laboratories39 indicated that changes in the quantity of TnI bound to the thin filament produce dramatic shifts in the pCa-force relation to either higher or lower pCa50 values (pCa50 is the -log calcium concentration required to induce 50% of the maximum Ca2+-dependent force). Moreover, myofilament control mechanisms may be quite sensitive to effects of proteolytic products. In fact, the addition of as little as four N-terminal peptides of MLC1 per thin filament can dramatically change the response of the myofilaments to Ca2+.19 Although there are conflicting reports on the nature and extent of myofilament dysfunction with different degrees of ischemia/reperfusion injury, the present study is the first to correlate the severity of ischemia/reperfusion injury with both an increase in myofilament dysfunction and the extent of myofilament protein degradation or loss.
Changes in Myofilament Proteins Induced by
Ischemia/Reperfusion
Earlier studies have also implicated troponin, or one of its
subunits, as a site for lesions associated with myocardial
ischemia/reperfusioninduced injury. In a study of dog hearts,
Toyo-Oka and Ross10 reported a loss of TnI from
myofibrils isolated from myocardial tissue made ischemic in
situ. A loss of TnI was also suggested by the results of Andres et
al,40 on the basis of the response to
isoproterenol of isolated rat hearts that experienced 20 minutes of
ischemia followed by reperfusion. These myofibrils demonstrated
an increase in Ca2+ sensitivity compared with
preparations from control hearts. We previously provided evidence that
myofibrils from isolated rat hearts made globally ischemic for
60 minutes contain degradation products of both TnI and
TnT.11 Although a TnI degradation product was
not confirmed in a subsequent study,9 we have
shown in the present study that TnI is degraded during global
ischemia (Fig 6
). Furthermore, the recent results reported by
Gao et al,7 together with data in the present
study, confirm our earlier conclusion that TnI degradation is an
important feature of ischemic damage. We have identified the
TnI degradation product using two different anti-TnI antibodies
(Mab E2 and 3309) that recognize different amino acid sequences of TnI.
This eliminates the possibility of nonspecific cross-reactivity with
proteins other than TnI.
An important aspect of the results presented here is that the
degradation product of TnI, which we believe is responsible in part
for the functional changes, remains bound to the myofilaments under
severe conditions, such as detergent treatment. In contrast to the case
with
-actinin, MLC1 (as well as MLC1 fragment 20 to 199), Tm, and
TnT, all of which appeared in the reperfusion effluent after 60 minutes
of ischemia, neither TnI nor TnI degradation products were
detected in the same effluent. This indicates that the TnI degradation
products are likely to have a role in the development of the
myofilament dysfunction, especially since degradation progresses from
one to two degradation products as the time of ischemia
increases.
The tightly bound degradation products arise from the hydrolysis at
the C-terminus of TnI. This proposal is based on our inability to
sequence the product, which is undoubtedly due to a blocked
N-terminus and the weak binding of Mab 10F2 to the TnI degradation
product compared with either Mab E2 or 3309. The Mab 10F2 epitope
lies between amino acid residues 188 and 199 (Fig 7
), indicating that
the degradation product is missing this C-terminal region of TnI.
TnI is a key protein in the Ca2+ switch
activating cardiac myofilaments.41 In
diastole, the actin-myosin reaction is believed to be
inhibited in part by tight binding of TnI to actin-Tm.
Ca2+ binding to the thin-filament receptor TnC
releases the inhibition by promoting tight binding of TnI to TnC with
Ca2+ bound. Using various C-terminal TnI
truncated mutants, recent work in our laboratory has systematically
investigated the structure-function relations of this region.
Interestingly, removal of C-terminal amino acids indicates that it is
this region of TnI that is key to this switching from actin to TnC
binding.23 It would therefore be expected that
proteolytic cleavage of C-terminal amino acids of TnI would result in
alterations in myofilament activation.
Loss of Fmax and a change in sensitivity to
Ca2+ could also result from either the loss of
MLC1 or
-actinin.
-Actinin, a main component of the Z lines,
forms an antiparallel dimer with actin binding sites located within the
N-terminus and is thought to link the thin filaments together.
Schroeter et al42 proposed that the Z line may
undergo dynamic changes during the crossbridge cycle and may be
important in keeping the thin filaments in register during
contraction/relaxation cycles. The loss of
-actinin (and possibly
other cytoskeletal proteins) has been previously proposed to be
responsible for reduced rigor or maximum
tension.12 43 Possible consequences of the loss
of
-actinin and unraveling of the Z line that could be associated
with a reduced maximum force include (1) ineffective transmission of
the force from the actin-myosin reaction to the ends of the sarcomere
and (2) altered interfilament spacing reducing the probability of
crossbridges reacting with the thin filament. Whether the degradation
of
-actinin observed in left ventricular tissue obtained
from hearts exposed to 60 minutes of ischemia with no
reperfusion is responsible for the loss of
-actinin from the
myofilament is not clear. The affinity of
-actinin for actin, which
may decrease if degraded, alters the type of network formed (parallel
filament versus isotropic networks).44
Reduced actin-myosin affinity and myosin ATPase activity have been
reported for cardiac myosin containing an MLC1 fragment that was
proteolysed at its N-terminus.45 This suggested
that MLC1 fragment 20 to 199 could alter function. However, since the
MLC fragment is released from the cell during severe ischemia
(Fig 3
), it would make only a small contribution toward the myofilament
dysfunction observed with 60 minutes of ischemia and 45 minutes
of reperfusion. Most likely, the loss of function under these severe
conditions is due to the massive loss of cell membrane integrity and
the release of cytoplasm and many myofilament proteins from the
cell.
Our results extend previous data showing that cellular proteins not
normally associated with contractile proteins bind to myofilaments
during ischemia/reperfusion. GAPDH, a cytosolic protein, seems
to associate with the actin filament under ischemic
conditions.9 It has been previously reported that another
cytoplasmic protein,
/ß crystallin, also may associate with the
myofilament under ischemic or anoxic
conditions.8 9 We have confirmed the presence of
these proteins in tissue obtained from hearts that experienced 15 or 60
minutes of ischemia/reperfusion as well as in the reperfusion
effluent after 60 minutes of ischemia, but not in control
hearts. In addition to these proteins, we detected ATP synthase
chain and OSC proteins. These proteins are localized on the cytoplasmic
side of the plasma membrane of the ATPase synthase complex located in
the inner membrane of the mitochondria. Earlier work showed that when
anoxic cultured heart cells are reoxygenated, there is a
release of proteins situated on the outer membrane of the
mitochondria.46 Blood of patients with myocardial
infarction show release of mitochondrial
proteins.47 48 49 Our data shed new light on this
release and indicate that two effects may result: a loss of ATP
synthetic capacity and a potential alteration in the myofilaments as
these products associate with the contractile proteins.
Substantiation of this latter speculation requires systematic
investigation of the effects of the ATP synthase proteins on
myofilament activity and regulation.
Mechanisms for Changes in Myofilament Proteins
Degradation and loss of some of these proteins may be attributable
to Ca2+-dependent proteases, which have been
proposed to be activated during the Ca2+
overload during late ischemia and early
reperfusion.43 50 One such
Ca2+-dependent protease thought to play a role in
ischemia is calpain,7 12 13 14 which is
localized near the Z line.51 In vitro,
-actinin, spectrin, desmin,52 53 54 55 TnI, and
TnT50 are susceptible to degradation by calpain.
Functional protection occurs by addition of a calpain
inhibitor during reperfusion of an isolated heart following
ischemia.15 16 The addition of calpain to
skinned muscle fibers causes a decrease in rigor tension with a
concurrent loss of a 95-kD protein.43 This
protein is probably
-actinin because of its molecular weight and
because the addition of calpain to myofibrils or skinned muscle fibers
results in the loss of the Z line (which is primarily composed of
-actinin43 55 ). In addition to the loss of
-actinin, Gao et al7 recently showed that
addition of calpain to skinned muscle fibers yields the same or similar
TnI degradation product as mild ischemia/reperfusion. Thus,
exogenous calpain mimics the alterations observed in the myofilament
proteins, ie, loss of
-actinin and degradation of TnI at the
C-terminus during mild ischemia. Whether other proteases, such
as mekratin,56 are involved remains an open
question.
The present study clearly shows that the contractile proteins TnI
and
-actinin are highly susceptible to progressive damage during
ischemia and on reperfusion. The alterations in TnI and
-actinin provide a plausible mechanism for the dysfunction observed
in skinned trabecular muscle fibers from isolated hearts
that have experienced ischemia with and without reperfusion.
Understanding the consequences of these changes to the myofilament
proteins is critical for the development of new diagnostic
strategies and new therapies for the protection and treatment of
ischemia/reperfusion injury.
| Selected Abbreviations and Acronyms |
|---|
|
| Acknowledgments |
|---|
| Footnotes |
|---|
Received July 8, 1997; accepted October 31, 1997.
| References |
|---|
|
|
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J. van der Velden, D. Merkus, B.R. Klarenbeek, A.T. James, N.M. Boontje, D.H.W. Dekkers, G.J.M. Stienen, J.M.J. Lamers, and D.J. Duncker Alterations in Myofilament Function Contribute to Left Ventricular Dysfunction in Pigs Early After Myocardial Infarction Circ. Res., November 26, 2004; 95(11): e85 - e95. [Abstract] [Full Text] |
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B. S. Palmer, P. F. Klawitter, P. J. Reiser, and M. G. Angelos Degradation of rat cardiac troponin I during ischemia independent of reperfusion Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1269 - H1275. [Abstract] [Full Text] [PDF] |
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V. L.J.L. Thijssen, J. Ausma, L. Gorza, H. M.W. van der Velden, M. A. Allessie, I. C. Van Gelder, M. Borgers, and G. J.J.M. van Eys Troponin I Isoform Expression in Human and Experimental Atrial Fibrillation Circulation, August 17, 2004; 110(7): 770 - 775. [Abstract] [Full Text] [PDF] |
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D. A Colantonio, J. E Van Eyk, and K. Przyklenk Stunned peri-infarct canine myocardium is characterized by degradation of troponin T, not troponin I Cardiovasc Res, August 1, 2004; 63(2): 217 - 225. [Abstract] [Full Text] [PDF] |
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K. K. Parker, J. A. Lavelle, L. K. Taylor, Z. Wang, and D. E. Hansen Stretch-induced ventricular arrhythmias during acute ischemia and reperfusion J Appl Physiol, July 1, 2004; 97(1): 377 - 383. [Abstract] [Full Text] [PDF] |
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D. G. Soergel, D. Georgakopoulos, L. B. Stull, D. A. Kass, and A. M. Murphy Augmented systolic response to the calcium sensitizer EMD-57033 in a transgenic model with troponin I truncation Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1785 - H1792. [Abstract] [Full Text] [PDF] |
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S. B. Marston and C. S. Redwood Modulation of Thin Filament Activation by Breakdown or Isoform Switching of Thin Filament Proteins: Physiological and Pathological Implications Circ. Res., December 12, 2003; 93(12): 1170 - 1178. [Abstract] [Full Text] [PDF] |
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D. B. Foster, T. Noguchi, P. VanBuren, A. M. Murphy, and J. E. Van Eyk C-Terminal Truncation of Cardiac Troponin I Causes Divergent Effects on ATPase and Force: Implications for the Pathophysiology of Myocardial Stunning Circ. Res., November 14, 2003; 93(10): 917 - 924. [Abstract] [Full Text] [PDF] |
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C.-H. Huang, S. F. Vatner, A. P. Peppas, G. Yang, and R. K. Kudej Cardiac Nerves Affect Myocardial Stunning Through Reactive Oxygen and Nitric Oxide Mechanisms Circ. Res., October 31, 2003; 93(9): 866 - 873. [Abstract] [Full Text] [PDF] |
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A. van der Laarse Hypothesis: troponin degradation is one of the factors responsible for deterioration of left ventricular function in heart failure Cardiovasc Res, October 1, 2002; 56(1): 8 - 14. [Abstract] [Full Text] [PDF] |
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N. Buscemi, D. B. Foster, I. Neverova, and J. E. Van Eyk p21-Activated Kinase Increases the Calcium Sensitivity of Rat Triton-Skinned Cardiac Muscle Fiber Bundles via a Mechanism Potentially Involving Novel Phosphorylation of Troponin I Circ. Res., September 20, 2002; 91(6): 509 - 516. [Abstract] [Full Text] [PDF] |
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W. Wang, C. J. Schulze, W. L. Suarez-Pinzon, J. R.B. Dyck, G. Sawicki, and R. Schulz Intracellular Action of Matrix Metalloproteinase-2 Accounts for Acute Myocardial Ischemia and Reperfusion Injury Circulation, September 17, 2002; 106(12): 1543 - 1549. [Abstract] [Full Text] [PDF] |
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O. Zeitz, A. E. Maass, P. Van Nguyen, G. Hensmann, H. Kogler, K. Moller, G. Hasenfuss, and P. M.L. Janssen Hydroxyl Radical-Induced Acute Diastolic Dysfunction Is Due to Calcium Overload via Reverse-Mode Na+-Ca2+ Exchange Circ. Res., May 17, 2002; 90(9): 988 - 995. [Abstract] [Full Text] [PDF] |
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C. Communal, M. Sumandea, P. de Tombe, J. Narula, R. J. Solaro, and R. J. Hajjar Functional consequences of caspase activation in cardiac myocytes PNAS, April 30, 2002; 99(9): 6252 - 6256. [Abstract] [Full Text] [PDF] |
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D. A. Colantonio, W. Pickett, R. J. Brison, C. E. Collier, and J. E. Van Eyk Detection of Cardiac Troponin I Early after Onset of Chest Pain in Six Patients Clin. Chem., April 1, 2002; 48(4): 668 - 671. [Full Text] [PDF] |
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H. Ruetten, C. Badorff, C. Ihling, A. M. Zeiher, and S. Dimmeler Inhibition of caspase-3 improves contractile recovery of stunned myocardium, independent of apoptosis-inhibitory effects J. Am. Coll. Cardiol., December 1, 2001; 38(7): 2063 - 2070. [Abstract] [Full Text] [PDF] |
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B. M Wolska, K. Vijayan, G. M Arteaga, J. P Konhilas, R. M Phillips, R. Kim, T. Naya, J. M Leiden, A. F Martin, P. P de Tombe, et al. Expression of slow skeletal troponin I in adult transgenic mouse heart muscle reduces the force decline observed during acidic conditions J. Physiol., November 1, 2001; 536(3): 863 - 870. [Abstract] [Full Text] [PDF] |
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N. Stupka and P. M. Tiidus Effects of ovariectomy and estrogen on ischemia-reperfusion injury in hindlimbs of female rats J Appl Physiol, October 1, 2001; 91(4): 1828 - 1835. [Abstract] [Full Text] [PDF] |
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D. K. Arrell, I. Neverova, and J. E. Van Eyk Cardiovascular Proteomics : Evolution and Potential Circ. Res., April 27, 2001; 88(8): 763 - 773. [Abstract] [Full Text] [PDF] |
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J. L. McDonough, R. Labugger, W. Pickett, M. Y. Tse, S. MacKenzie, S. C. Pang, D. Atar, G. Ropchan, and J. E. Van Eyk Cardiac Troponin I Is Modified in the Myocardium of Bypass Patients Circulation, January 2, 2001; 103(1): 58 - 64. [Abstract] [Full Text] [PDF] |
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Y. G. Wang, W. J. Benedict, J. Huser, A. M. Samarel, L. A. Blatter, and S. L. Lipsius Brief rapid pacing depresses contractile function via Ca2+/PKC-dependent signaling in cat ventricular myocytes Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H90 - H98. [Abstract] [Full Text] [PDF] |
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J. James, Y. Zhang, H. Osinska, A. Sanbe, R. Klevitsky, T. E. Hewett, and J. Robbins Transgenic Modeling of a Cardiac Troponin I Mutation Linked to Familial Hypertrophic Cardiomyopathy Circ. Res., October 27, 2000; 87(9): 805 - 811. [Abstract] [Full Text] [PDF] |
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R. Labugger, L. Organ, C. Collier, D. Atar, and J. E. Van Eyk Extensive Troponin I and T Modification Detected in Serum From Patients With Acute Myocardial Infarction Circulation, September 12, 2000; 102(11): 1221 - 1226. [Abstract] [Full Text] [PDF] |
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A. J. Sherman, F. J. Klocke, R. S. Decker, M. L. Decker, K. A. Kozlowski, K. R. Harris, S. Hedjbeli, Y. Yaroshenko, S. Nakamura, M. A. Parker, et al. Myofibrillar disruption in hypocontractile myocardium showing perfusion-contraction matches and mismatches Am J Physiol Heart Circ Physiol, April 1, 2000; 278(4): H1320 - H1334. [Abstract] [Full Text] [PDF] |
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B. G. Fogelson, S. I. Nawas, and W. R. Law MECHANISMS OF MYOCARDIAL PROTECTION BY ADENOSINE-SUPPLEMENTED CARDIOPLEGIC SOLUTION: MYOFILAMENT AND METABOLIC RESPONSES J. Thorac. Cardiovasc. Surg., March 1, 2000; 119(3): 601 - 609. [Abstract] [Full Text] [PDF] |
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L Rappaport Ischemia-reperfusion associated myocardial contractile dysfunction may depend on Ca2+-activated cytoskeleton protein degradation Cardiovasc Res, March 1, 2000; 45(4): 810 - 812. [Full Text] [PDF] |
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Z. Papp, J. van der Velden, and G.J.M Stienen Calpain-I induced alterations in the cytoskeletal structure and impaired mechanical properties of single myocytes of rat heart Cardiovasc Res, March 1, 2000; 45(4): 981 - 993. [Abstract] [Full Text] [PDF] |
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J. A. Simpson, J. E. van Eyk, and S. Iscoe Hypoxemia-induced modification of troponin I and T in canine diaphragm J Appl Physiol, February 1, 2000; 88(2): 753 - 760. [Abstract] [Full Text] [PDF] |
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A. M. Murphy, H. Kögler, D. Georgakopoulos, J. L. McDonough, D. A. Kass, J. E. Van Eyk, and E. Marbán Transgenic Mouse Model of Stunned Myocardium Science, January 21, 2000; 287(5452): 488 - 491. [Abstract] [Full Text] |
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S. A. Thomas, J. A. Fallavollita, T.-C. Lee, J. Feng, and J. M. Canty Jr Absence of Troponin I Degradation or Altered Sarcoplasmic Reticulum Uptake Protein Expression After Reversible Ischemia in Swine Circ. Res., September 3, 1999; 85(5): 446 - 456. [Abstract] [Full Text] [PDF] |
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D. B. Foster and J. E. Van Eyk In Search of the Proteins That Cause Myocardial Stunning Circ. Res., September 3, 1999; 85(5): 470 - 472. [Full Text] [PDF] |
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Q. Shi, M. Ling, X. Zhang, M. Zhang, L. Kadijevic, S. Liu, and J. P. Laurino Degradation of Cardiac Troponin I in Serum Complicates Comparisons of Cardiac Troponin I Assays Clin. Chem., July 1, 1999; 45(7): 1018 - 1025. [Abstract] [Full Text] [PDF] |
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J. M.J. Lamers Preconditioning and limitation of stunning: one step closer to the protected protein(s)? Cardiovasc Res, June 1, 1999; 42(3): 571 - 575. [Full Text] [PDF] |
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N. G. Perez, E. Marban, and H. E Cingolani Preservation of myofilament calcium responsiveness underlies protection against myocardial stunning by ischemic preconditioning Cardiovasc Res, June 1, 1999; 42(3): 636 - 643. [Abstract] [Full Text] [PDF] |
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J. E. Saffitz, R. B. Schuessler, and K. A. Yamada Mechanisms of remodeling of gap junction distributions and the development of anatomic substrates of arrhythmias Cardiovasc Res, May 1, 1999; 42(2): 309 - 317. [Full Text] [PDF] |
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R. Bolli and E. Marban Molecular and Cellular Mechanisms of Myocardial Stunning Physiol Rev, April 1, 1999; 79(2): 609 - 634. [Abstract] [Full Text] [PDF] |
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J. L. McDonough, D. K. Arrell, and J. E. Van Eyk Troponin I Degradation and Covalent Complex Formation Accompanies Myocardial Ischemia/Reperfusion Injury Circ. Res., January 22, 1999; 84(1): 9 - 20. [Abstract] [Full Text] [PDF] |
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R. J. Solaro Troponin I, Stunning, Hypertrophy, and Failure of the Heart Circ. Res., January 22, 1999; 84(1): 122 - 124. [Full Text] [PDF] |
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A. G. Katrukha, A. V. Bereznikova, V. L. Filatov, T. V. Esakova, O. V. Kolosova, K. Pettersson, T. Lovgren, T. V. Bulargina, I. R. Trifonov, N. A. Gratsiansky, et al. Degradation of cardiac troponin I: implication for reliable immunodetection Clin. Chem., December 1, 1998; 44(12): 2433 - 2440. [Abstract] [Full Text] [PDF] |
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J. E. Van Eyk, D. K. Arrell, D. B. Foster, J. D. Strauss, T. Y. K. Heinonen, E. Furmaniak-Kazmierczak, G. P. Cote, and A. S. Mak Different Molecular Mechanisms for Rho Family GTPase-dependent, Ca2+-independent Contraction of Smooth Muscle J. Biol. Chem., September 4, 1998; 273(36): 23433 - 23439. [Abstract] [Full Text] [PDF] |
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N. G. Perez, W. D. Gao, and E. Marban Novel Myofilament Ca2+-Sensitizing Property of Xanthine Oxidase Inhibitors Circ. Res., August 24, 1998; 83(4): 423 - 430. [Abstract] [Full Text] [PDF] |
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C. Communal, M. Sumandea, P. de Tombe, J. Narula, R. J. Solaro, and R. J. Hajjar Functional consequences of caspase activation in cardiac myocytes PNAS, April 30, 2002; 99(9): 6252 - 6256. [Abstract] [Full Text] [PDF] |
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S.-J. Kim, R. K. Kudej, A. Yatani, Y.-K. Kim, G. Takagi, R. Honda, D. A. Colantonio, J. E. Van Eyk, D. E. Vatner, R. L. Rasmusson, et al. A Novel Mechanism for Myocardial Stunning Involving Impaired Ca2+ Handling Circ. Res., October 26, 2001; 89(9): 831 - 837. [Abstract] [Full Text] [PDF] |
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