© 1996 American Heart Association, Inc.
Articles |
Binding of Cytosolic Proteins to Myofibrils in Ischemic Rat Hearts
Correspondence to Prof Fabio Di Lisa, Dipartimento di Chimica Biologica, Via Trieste, 75, 35121 Padova, Italy. E-mail dilisa@civ.bio.unipd.it.
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Abstract |
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Abstract Myofibrillar proteins (MPs) were extracted from isolated and perfused rat hearts subjected to different periods of ischemia to investigate the occurrence of protein degradation and/or the association of cytosolic proteins with the myofibrillar pellet. A 23-kD band was detected by SDS-PAGE of MPs after 5 minutes of ischemia, with its density gradually increasing to a plateau after 20 minutes. Longer periods of ischemia were associated with the appearance of a 39-kD band. Irrespective of the duration of ischemia, both these bands persisted during reperfusion. A partial proteolytic degradation of troponin T (TnT) and troponin I (TnI) has been claimed to be responsible for the generation of these peptides. However, the N-terminal sequence of the 39-kD band was identical to that of GAPDH, whereas Edman sequencing after pepsin digestion showed that the 23 kD is
B-crystallin. The binding of the
two cytosolic proteins to myofibrils was confirmed by
immunofluorescence analysis on cryosections
of ischemic hearts. In vitro studies showed that acidosis was
sufficient to induce the binding of B-crystallin, whereas the
inhibition of ATP depletion prevented the binding of GAPDH. Thiol
oxidation is unlikely to promote GAPDH binding, since perfusion with
iodoacetate under aerobic conditions or treatment of
homogenates with N-ethylmaleimide or diamide
failed to induce GAPDH association with the myofibrils. These changes
of the myofibrillar proteins could be considered as intracellular
markers of the evolution of the ischemic damage. In addition,
the binding of the 23-kD peptide might be involved in alterations of
contractility.
Key Words: GAPDH • B-crystallin • acidosis • troponin • ischemia
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During ischemia, proteolysis results in ultrastructural alterations, which are among the major causes of irreversible injury of the myocardium.1 2 3 Less defined is the contribution of proteolysis to the contractile impairment that is produced when reperfusion takes place after a short period of ischemia. This functional abnormality, which has been defined as myocardial stunning4 and occurs in the absence of any ultrastructural sign of necrosis, does not seem to be caused by metabolic alterations.5 In a recent review, oxyradicals, acidosis, accumulation of metabolites, and impairment of energy production were ruled out as possible causes of stunning, leaving as a likely mechanism the decrease of myofibrillar affinity for Ca2+ resulting from covalent or degradative processes affecting the myofibrillar proteins.5 This hypothesis has been supported by a recent report on the TnI and TnT degradation induced by autolysis in rat hearts.6 The major evidence of troponin fragmentation was represented by the appearance of two peptides that were stained by anti-troponin antibodies in immunoblots. However, since cross-reactivities with other proteins were not taken into account and the identity of the peptides was not confirmed by their amino acid sequence, other mechanisms could be involved in the modifications of the myofibrillar pellet associated with autolysis. During anoxic conditions, the drastic changes in pH and ionic concentrations could modify the intracellular distribution of proteins. In many tissues, the alterations of the intracellular milieu are reflected by the binding of cytosolic proteins, such as glycolytic enzymes, to membrane or cytoskeletal proteins.7 8 9 10 11 12 13 14 15 16 Recently,
B-crystallin, a
constitutive cytosolic protein of cardiac myocytes, has been shown to
bind to myofibrils during ischemia.17 Thus, a
false-positive reaction of antibodies against myofibrillar
components with other proteins that are not associated with myofibrils
under physiological conditions could be erroneously
interpreted as evidence of protein degradation.
We have investigated the occurrence of protein degradation or the
association of cytosolic proteins with the myofibrillar pellet in
isolated and perfused rat hearts subjected to different periods of
global ischemia. Our results indicate that the two bands that
were previously indicated as troponin fragments are GAPDH and
B-crystallin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolated Heart Protocols
Adult male Wistar rats (180 to 200 g) were used. Isolated hearts were perfused by the nonrecirculating Langendorff technique, as previously described.18 The substrates were either 11 mmol/L glucose or 1 mmol/L pyruvate when GAPDH was inhibited by 0.15 mmol/L iodoacetate. After a 20-minute equilibration, hearts were either perfused under normoxic conditions for 60 minutes or made ischemic by stopping the coronary flow for periods ranging from 5 to 60 minutes. In another group of hearts, ischemia was followed by 30 minutes of reperfusion. In a separate set of experiments, Ca2+ was substituted with 0.25 mmol/L EGTA in the perfusion medium. In this case, a 30-minute equilibration was followed by another 30-minute period either in the presence or absence of 1 µmol/L FCCP. Autolysis was performed by incubating freshly excised hearts in impermeable plastic bags at 37°C.6 The perfusion or the autolysis protocols were terminated by clamping the hearts with tongues precooled in liquid nitrogen.
To obtain an isovolumetrically beating preparation, a saline-filled
latex balloon, connected via a catheter to a Statham transducer (P
2306), was inserted into the left ventricle through an atriotomy and
secured with suture around the atrioventricular
groove.18 The balloon was inflated to provide an
end-diastolic pressure of 
5.0 mm Hg. Two
three-way stopcocks between the transducer, syringe, and the tube
leading to the heart allowed for continuous monitoring of left
ventricular developed pressure recorded by a
Hewlett-Packard HP7754A thermal tip recorder.
In Vitro Protocols
The whole homogenate (0.5 g/30 mL) obtained from
freshly excised hearts was treated by incubation under different
conditions of pH (7.2 and 6.9) and temperature (4°C and 37°C) for
15 minutes. In other incubation protocols performed at pH 7.2 and
37°C, the homogenate was added with one of the following
sets of compounds: (1) 10 mmol/L creatine phosphate and 5 mmol/L
ATP/Mg2+, (2) 2 mmol/L EGTA, (3) 2 mmol/L diamide,
(4) 2 mmol/L NEM, and (5) 50 µmol/L
N-methylarginine.19 Each procedure was repeated
at least four times. At the end of these treatments, the
homogenates were centrifuged, and myofibrils were
extracted as described below.
Metabolite and Enzyme Assays
The tissue contents of ATP and CP were assayed as previously
described.18
Myofibril Extraction
Myofibrils were isolated in the presence of protease
inhibitors (0.5 µg/mL leupeptin, 0.5 µg/mL pepstatin A,
and 0.2 mmol/L phenylmethylsulfonyl fluoride) as previously
described.6 20 The entire procedure was performed at
4°C. Briefly, 0.5 to 0.6 g of frozen ventricles was
homogenized in 30 vol of 20 mmol/L imidazole, pH 7.0, and
mixed with a further 30 vol of solution A consisting of 60 mmol/L KCl,
30 mmol/L imidazole, pH 7.0, and 2.5 mmol/L MgCl2. This
suspension was centrifuged at 10 000g for 15
minutes, and the resulting pellet was resuspended to the original
homogenate volume in solution A. After a second
centrifugation at 4000g for 10 minutes, the
pellet was suspended again in solution A containing 2 mmol/L EGTA. The
pellet resulting from a further centrifugation at
4000g for 10 minutes was added with solution A containing
1% Triton X-100 and homogenized with a Teflon-glass
hand homogenizer. The resulting suspension was
subjected to three cycles of centrifugation,
resuspension, and homogenization using solution A
devoid of MgCl2 (solution B) to remove Triton X-100. The
final pellet was resuspended in 2 mL of solution B. This isolation
procedure proved to be essential to eliminate visible contaminating
proteins in the regions of 23 and 39 kD in the electrophoreses of
myofibrils from control normoxic hearts.
SDS-PAGE and Immunoblotting
Protein concentration was determined as previously
described.21 Myofibrillar proteins were separated by
SDS-PAGE and stained with Coomassie Blue. Densitometry was
performed on scanned gels by using the IPLab Gel computer program for
the MacIntosh (Signal Analytics Co). The intensities of the 23- and
39-kD bands were normalized to the tropomyosin band in the same gel
lane. GAPDH binding was calculated by comparison with a standard curve
generated by loading various amounts of purified GAPDH
(Boehringer Mannheim). Duplicate gels were electrophoretically
transferred to either nitrocellulose sheets22 or PVDF,
which ameliorated the transfer of the 39-kD band. Blots incubated with
the following antibodies were used for immunoblotting
analyses: (1) anti-TnT mAbs: RV-C223 and JLT-12
(Sigma Chemical Co); (2) TnI mAbs: TI-1, TI-3, and TI-4,24
and four other TnI mAbs (L. Saggin and S. Schiaffino, unpublished
clones); (3) GAPDH mAb 6G5 (Sigma); and (4) anti–B-crystallin
pAb,17 a generous gift from Dr M. Chiesi. Bound antibodies
were revealed by anti-mouse or anti-rabbit immunoglobulin
conjugated with horseradish peroxidase or alkaline phosphatase.
Immunofluorescence
Indirect immunofluorescence was performed as
previously described.25 Cryosections (
7 µm thick)
from frozen ventricles were washed with PBS for 15 minutes to remove
cytosolic proteins. Cryosections were then incubated with adequate
dilutions of either GAPDH mAb or B-crystallin pAbs for 20 minutes at
37°C. The antibodies were diluted in PBS containing 0.3% BSA. After
several rinses with PBS, sections were incubated with
rhodamine-conjugated secondary antibodies, washed again in PBS,
mounted with glycerol, and observed with a Zeiss Axioplan microscope
equipped with epifluorescence optics.
For double-labeling immunofluorescence, antibody staining was followed by a further incubation with fluorescein-conjugated phalloidin, which binds to actin.
N-Terminal Sequencing
For N-terminal sequencing analysis, the isolated
myofibrils were separated in an SDS-PAGE (12.5%) according to the
Laemmli system26 and electroblotted onto a PVDF membrane
using the Towbin buffer.22
After transfer, the membrane sheet was extensively rinsed with water and stained in 0.1% Coomassie Blue in 50% methanol/water. Destaining was performed in 50% methanol/water and, finally, water. Sequencing of the excised bands was carried out using an Applied Biosystem 476A sequencer with on-line PTH detection.
Pepsin Digestion of the 23-kD Protein
Isolated myofibrils were separated on a Laemmli gel (12.5%),
and the bands corresponding to the 23-kD protein were excised from
several lanes after Coomassie staining. The bands were washed several
times in distilled water and incubated overnight in the gel loading
buffer. The material was then applied to an SDS-PAGE composed of a 4%
stacking and a 9% resolving gel, according to the method of
Laemmli.26 The concentrated sample was electroblotted onto
a PVDF membrane as described before. After Coomassie staining, the
protein band was cut in 1- to 2-mm squares, and the Coomassie was
eliminated by a 2-minute wash in 70% acetonitrile. The pepsin
digestion was performed by covering the pieces of membrane with 70%
formic acid and by addition of 1 µg of pepsin. After 3 hours of
incubation at room temperature, the digestion mixture was resolved by
reversed-phase HPLC using an Aquapore RP300 (100x1 mm, 7 µm).
The buffers used were as follows: A, 0.1% trifluoroacetic acid; B,
0.08% trifluoroacetic acid and 70% acetonitrile.
Trypsin Digestion
The protein of interest was concentrated and electroblotted onto
a PVDF membrane as described for pepsin digestion. The in situ
digestion of the blotted protein was performed by covering the excised
band with 10 µL of 25 mmol/L ammonium bicarbonate, 1%
ß-d-octylglucopyranoside, 10% methanol, and 0.1 µg/µL
trypsin (Promega modified sequence grade). After 18 hours of
incubation, formic acid was added to a final concentration of 50% to
allow release of peptides bound to the membrane. The extraction with
formic acid was repeated a second time.
Mass Profile Fingerprinting
For mass fingerprinting analysis, an aliquot of the
digestion mixture was injected onto a reversed-phase capillary HPLC
column (C18, 5 µm, 300A) directly connected to a triple quadrupole
mass spectrometer (model TSQ700, Finnigan Mat) equipped with an
electrospray ionization source.27
The search was performed by generating the mass profile of the protein of interest by digestion with trypsin, followed by the determination of the masses of the fragments produced. The resulting mass fingerprint was used to search a database in which all the protein sequences are replaced by the theoretical mass fingerprints produced by trypsin digestion.27
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two major changes were detected when the electrophoretic profile of myofibrils extracted from normoxic hearts was compared with that of myofibrils from ischemic hearts (Fig 1
). A
23-kD band was detected in myofibrils extracted from hearts made
ischemic for 5 minutes: its staining intensity increased
gradually, reaching a plateau when the duration of ischemia
exceeded 20 minutes (Fig 1 and Table). Longer periods of
ischemia, ie, >30 minutes, were characterized by the
appearance of a 39-kD band along with a drastic reduction of tissue ATP
contents. Irrespective of the duration of ischemia, both bands
persisted during postischemic reperfusion (Fig 1 and
Table). Both the 23- and the 39-kD bands were also detected in the
myofibrillar pellet prepared from hearts subjected to 60 minutes of
autolysis, confirming similar recent findings by Westfall and
Solaro.6 On the basis of their interpretation, we
initially investigated the 23- and the 39-kD bands by
immunoblotting and amino acid sequencing as possible
fragments of TnT and TnI. Under the electrophoretic conditions adopted
for this study, a single TnT band (41 kD) was visualized in
Western blots probed with both RV-C2 and JLT-12; the TnT mAb also used
by Westfall and Solaro6 (Fig 2A). The 39-kD
band present in myofibrils from ischemic hearts failed to
react with RV-C2 but was positive with JLT-12 (Fig 2A). The fact that
the 39-kD band was not a TnT fragment was established by partial
N-terminal sequencing: the sequence obtained was instead VKVGVNGFG,
which was identical to that of the N-terminal domain of GAPDH. The
identity with GAPDH was conclusively established by mass fingerprint
analysis, which also excluded the presence of other proteins
comigrating with the 39-kD band. The trypsin digestion of the 39-kD
band produced seven fragments with the following weights: 595.5, 643,
665, 739, 869, 1227.5, and 1369.5. The highest score27 of
this mass fingerprint was for mammalian GAPDH from three different
species, namely, rat, mouse, and Chinese hamster. This unambiguously
defined the 39-kD protein as GAPDH. Considerably lower scores were
given from other sources (avian or pig) or still other proteins
(cytoplasmic trehalase and glutamine synthetase adenyltransferase from
Escherichia coli and transcription-repair coupling
factor from Bacillus subtilis). To confirm these results,
the 39-kD band was probed in Western blots with a GAPDH mAb (Fig 2B).
Myofibrils extracted from hearts either perfused under normoxic
conditions or made ischemic for <30 minutes failed to react
with the antibody, whereas an evident band, corresponding to the 39-kD
peptide, showed up in samples from hearts subjected to prolonged
ischemia or autolysis (Fig 2B).
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The 23-kD band was not labeled by the seven TnI mAbs in
immunoblotting. The results obtained with three of them
are shown in Fig 3A. The band was not recognized by any
TnT mAb tested either (not shown). Conclusive demonstration that this
peptide was not a TnI fragment was provided by Edman sequencing of a
peptide (TVNGPRKQASGPERTIP) produced by pepsin digestion. This
analysis clearly indicated that the 23-kD peptide is
B-crystallin, confirming a previous report17 that
showed the disappearance of B-crystallin from cytosolic fractions of
ischemic hearts. Immunoblots of myofibrils
extracted from aerobic hearts and stained by B-crystallin pAb (Fig 3B) did not react, whereas a single band, corresponding to the 23-kD
peptide, was present in samples from ischemic hearts. This
immunoreactivity was used to study the movements of B-crystallin
from the cytosol to the myofibrils (Fig 4B): the protein
had completely disappeared from the cytosol after 60 minutes of
ischemia, whereas 13.5±5.8% of the total cellular GAPDH
became bound to myofibrils upon ischemia (Fig 4A).
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The ischemia-induced association of GAPDH with myofibrils
was confirmed by immunofluorescence experiments:
soluble proteins were removed by washing the sections with PBS for 15
minutes before immunostaining. Normoxic heart sections
were not stained by the GAPDH mAb (Fig 5A), whereas foci
of reactive fibers were observed in sections of hearts made
ischemic for 30 minutes (Fig 5B and 5D). In addition,
striations were evident in longitudinal sections of ischemic
hearts, suggesting GAPDH binding to myofibrillar components (Fig 5C). A
similar pattern of immunoreactivity was observed with B-crystallin
pAb and was already present after shorter periods of
ischemia (10 minutes). Double
immunofluorescence analysis showed that
anti-GAPDH and anti–B-crystallin staining colocalized with
fluorescein-phalloidin staining corresponding to I-band
localization (not shown).
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The 23- or the 39-kD bands were also observed in the myofibrillar
preparation when normoxic hearts were deenergized by an uncoupler of
oxidative phosphorylation (FCCP). To study the possible
contribution of [Ca2+]i increase to the
myofibrillar binding of the 23- and 39-kD species, hearts were perfused
for 20 minutes with EGTA to deplete intracellular Ca2+
stores28 and to prevent the uptake of extracellular
Ca2+. When this treatment was protracted over another
30-minute period, the profile of the myofibrillar proteins was
identical to that of control hearts (Fig 6, lane 3),
indicating that asystole per se is not responsible for the binding.
Conversely, the addition of FCCP induced the appearance of the 23- and
the 39-kD bands (Fig 6, lane 4). These results rule out the involvement
of intracellular Ca2+ overload. In addition, the ratio of
NADH to NAD is not involved, since B-crystallin and GAPDH bind to
myofibrils both when the oxidized form is prevailing, such as upon FCCP
addition, and when NADH oxidation is prevented, such as during
ischemia.
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The effect of acidosis was then investigated by perfusing hearts with
hypercarbic buffers (pH 6.9), a condition that has been shown to
decrease pHi29 but not alter tissue ATP and CP
contents.30 This perfusion protocol resulted in the
binding of B-crystallin but not of GAPDH (Fig 6
, lane 6).
The oxidation of sulfhydryl groups, which has been reported to occur
during ischemia,31 is unlikely to promote
B-crystallin or GAPDH binding, since perfusion with iodoacetate
under aerobic conditions (Fig 6) or treatment of
homogenates with N-ethylmaleimide or diamide
failed to induce the association of these cytosolic proteins with the
myofibrils (results not shown).
The binding of both B-crystallin and GAPDH to myofibrils could be
reproduced in vitro by incubating the homogenate of a
normoxic heart at 37°C and pH <7.0 for 15 minutes (Fig 7, lane 2). However, heating and acidosis had different
effects on the binding of the two proteins. The incubation at 37°C
and pH 7.2, which made ATP undetectable, resulted in the binding of
only GAPDH (Fig 7, lane 3). On the other hand, when the whole
homogenate was incubated in the presence of 10 mmol/L CP,
ATP concentration was maintained in the millimolar range, and GAPDH
binding was totally prevented (Fig 7, lane 4). Conversely,
B-crystallin, but not GAPDH, was found in the myofibrillar pellet
when the incubation was performed at pH <7.0 and 4°C (Fig 7, lane
1). Thus, the association of one protein to the myofibrils is not
required to bind the other one.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Time-Dependent Changes of Myofibril Composition During Ischemia
The results of the present study show that two peptides with an apparent Mr of 23 and 39 kD appeared among the myofibrillar proteins of rat hearts subjected to either ischemia, autolysis, or metabolic inhibition (Figs 1
and 7). The 23- and 39-kD bands were identified as GAPDH and
B-crystallin, respectively. In addition, it can be excluded that the
appearance of these two peptides results from the degradation of other
myofibrillar proteins. Two peptides showing electrophoretic mobilities similar to those reported in the present study have already been observed in myofibrils isolated from autolyzed hearts.6 A partial proteolytic degradation of TnT and TnI has been claimed to be responsible for the generation of these peptides. This assertion was supported by the following observations: (1) a decrease in the density of TnT and TnI was determined by SDS-PAGE; (2) the peptide with the slower electrophoretic mobility was stained by a TnT mAb, whereas the faster one reacted with TnI pAb; and (3) this latter peptide could be phosphorylated by cAMP-dependent kinase (PKA) like TnI.32
It seems unlikely that the two peptides, which we characterized, are
different from the ones observed by Westfall and Solaro,6
since in the two studies the tissue source, the experimental model, and
the procedures for myofibrillar extraction and electrophoresis were
identical. The major variations concern the antibodies used to probe
the immunoblots. Westfall and Solaro used a TnT mAb
(JLT-12) and TnI pAb. We could reproduce the staining of the 39-kD band
using the JLT-12 TnT mAb (Fig 2A), but we failed to detect any labeling
of either the 39- or the 23-kD peptides when the Western blots were
probed with another TnT mAb and with a battery of TnI mAbs (Figs 2A and 3A). This apparent discrepancy can be solved by considering
false-positive reactions of some anti-troponin antibodies with
other proteins. For instance, some anti-TnT antibodies have been shown
to cross-react with GAPDH,33 as can be expected from
partial sequence homology of these two proteins. Indeed, our results
clearly show that the 39-kD peptide is not a TnT fragment but is
GAPDH.
As far as the 23-kD peptide is concerned, the sequence
analysis, besides identifying it as B-crystallin, allows us
to exclude any structural relationship with either TnI or TnT. In
addition, since B-crystallin has also been described as a substrate
for cAMP-dependent protein kinase,34 the
phosphorylation of the 23-kD peptide6
cannot be considered as proof of its identity as a TnI fragment.
Our results support the recent evidence (provided by Bennardini et
al17 ) of B-crystallin mobilization induced by
ischemia. However, in this latter report, the disappearance
from the cytosolic fraction or the appearance in a crude myofibrillar
pellet became significant only after 30 minutes, which is the point at
which the mobilization of the 23-kD peptide reached a plateau in the
present study (Fig 1). Bennardini et al have shown, in their
myofibrillar preparations, that other peptides have migration rates
similar to those of B-crystallin. The consequent decrease of the
signal-to-noise ratio could explain why longer periods of
ischemia were necessary to detect appreciable amounts of
B-crystallin among the myofibrillar proteins.
Although we provided evidence that the 23- and the 39-kD bands are not
products of troponin degradation, we cannot exclude the possibility
that troponin or other myofibrillar components might undergo
proteolysis during ischemia. It has been shown by densitometric
analyses that TnI amounts decrease either after 60 minutes of
rat heart autolysis6 or 24 hours after coronary
ligation in dog hearts.35 Thus, it appears that TnI
digestion, if any, is concomitant with or follows the transition toward
irreversible damage. The appearance of B-crystallin occurs at a much
earlier stage, providing additional evidence that it is not generated
by TnI digestion, as was previously suggested by associating the
decrease in TnI density after 60 minutes of autolysis with the
appearance of a supposed TnI fragment, which is already present
after 30 minutes.6
The disappearance of TnI has not yet been validated by a convincing demonstration of TnI fragments. We failed to demonstrate such fragments even by running the electrophoreses in a tricine buffer (results not shown), a procedure that allows the detection of peptides with Mr values as low as 3 kD.36 The lack of TnI fragments might be explained if the digestion is catalyzed by calpain(s). TnI and TnT are indeed calpain substrates, and we have demonstrated that, in vitro, these two components (but not TnC) of the troponin complex are completely digested by µ-calpain without the appearance of any lower molecular weight fragment.37
Possible Mechanisms and Significance of Cytosolic Proteins Binding
to Myofibrils
Among the metabolic changes known to be induced by
ischemia, acidosis and ATP depletion appeared to be more
relevant to the movement of B-crystallin and GAPDH, respectively. In
perfused hearts, deenergization was associated with the binding of both
proteins to myofibrils, whereas only B-crystallin was bound when
normoxic hearts were perfused with hypercarbic buffers (pH 6.9). Redox
changes of pyridine nucleotides, sulfhydryl group
oxidation, or intracellular Ca2+ overload did not seem to
contribute to the movement of the two cytosolic proteins.
In vitro studies on homogenates confirmed the effectiveness
of acidosis to induce the binding of B-crystallin to myofibrils.
The relationship between ATP decrease and GAPDH binding suggested by ischemia and mitochondrial uncoupling in perfused hearts is supported by the results of in vitro experiments. Indeed, when ATP hydrolysis in the homogenates was reduced by incubation at low temperature or manipulating the medium composition, GAPDH binding was completely prevented. In particular, GAPDH was not found in the myofibrillar pellet when ATP depletion in the homogenate was prevented by a large CP availability.
The mobilization of GAPDH from cytosol to the myofibrils induced by ischemia has already been described in perfused rat hearts and in sheep hearts.14 The degree of binding was reported to be minimal, not only because the duration of ischemia was limited to 10 minutes but also because the binding was measured as the amount of enzymatic activity that was recovered from the pellet after its resuspension in a so-called stabilization buffer. In our experience, the binding is associated with the loss of enzymatic activity, whereas only the treatment of the pellet with 5 mol/L urea was followed by a partial recovery of the bound GAPDH in the supernatant (results not shown).
The relationship between GAPDH binding to other proteins and ATP hydrolysis is probably not unique to the heart. A decrease of ATP concentration has been reported to favor the binding of GAPDH to skeletal muscle F-actin38 or to tubulin,39 whereas Mg2+ seems to enhance GAPDH binding to the thin filament from beef muscle.15
It is tempting to speculate that the mobilized cytosolic proteins might
protect myofibrillar components. This concept fits quite well with
B-crystallin, which has been characterized as a molecular
chaperone40 and binds to myofibrils during the reversible
phase of ischemia. The binding of cytosolic proteins to
myofibrils could modify the mechanical activity of the contractile
elements, and it may be implicated in myocardial stunning.
The significance of GAPDH binding, which occurs at a time when the evolution of the ischemic damage has already shifted toward cell necrosis, remains to be elucidated. It should be pointed out that numerous nonglycolytic activities have been attributed to GAPDH, including DNA repair,41 helicase activity,42 nuclear tRNA export,43 mRNA turnover and translation,44 protein phosphorylation,45 triad junction formation,8 and endocytosis.46 Interestingly, GAPDH binding to RNA is inhibited by ATP.44
Finally, the changes of the myofibrillar proteins could be used as
intracellular markers in the evolution of ischemic damage. The
detection of B-crystallin would mark the onset of ischemia,
whereas severe damage would be indicated by GAPDH. In addition, the
definitive loss of viability is associated with the appearance of
high molecular weight peptides, which cross react with Tn mAb as a
result of myofibrillar protein cross-linking.47
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by CNR grants 93.02075.CT14 and 94.00337.CT14 and by a grant of the European Economic Community Concerted Action Contract No. BMH1-CT92-1171 to Dr Schiaffino.
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Footnotes |
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From the Dipartimento di Chimica Biologica e Centro per lo Studio delle Biomembrane (R.B., R.M., E.C., F.D.L.), CNR, and the Dipartimento di Scienze Biomediche e Centro Biologia e Fisiopatologia Muscolare (S.S.), CNR, Università di Padova, Padova, Italy, and the Laboratory for Biochemistry (P.D., E.C.), Swiss Federal Institute of Technology (ETH), Zürich, Switzerland.
Received June 23, 1995; accepted February 2, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Jennings RB, Murry CE, Steenbergen C, Reimer KA. Development of cell injury in sustained acute ischemia. Circulation. 1990;82(suppl II):II-2-II-12.
2.
Steenbergen C, Hill ML, Jennings RB.
Cytoskeletal damage during myocardial ischemia: changes in
vinculin immunofluorescence staining during total
in vitro ischemia in dog heart. Circ
Res. 1987;60:478-486.
3.
Iwai K, Hori M, Kitabatake A, Kurihara H, Uchida K,
Inoue M, Kamada K. Disruption of microtubules as an early sign
of irreversible ischemic injury: immunohistochemical study of
in situ canine hearts. Circ Res. 1990;67:694-706.
4.
Braunwald E, Kloner RA. The stunned
myocardium: prolonged, postischemic
ventricular dysfunction.
Circulation. 1982;66:1146-1149.
5. Kusuoka H, Marban E. Cellular mechanisms of myocardial stunning. Annu Rev Physiol. 1992;54:243-256. [Medline] [Order article via Infotrieve]
6.
Westfall MV, Solaro RJ. Alterations in
myofibrillar function and protein profiles after complete global
ischemia in rat hearts. Circ Res. 1992;70:302-313.
7.
Pierce GN, Philipson KD. Binding of glycolytic
enzymes to cardiac sarcolemmal and sarcoplasmic reticular
membranes. J Biol Chem. 1985;260:6862-6870.
8.
Caswell AH, Corbett AM. Interaction of
glyceraldehyde-3-phosphate dehydrogenase with isolated
microsomal subfractions of skeletal muscle. J
Biol Chem. 1985;260:6892-6898.
9. Arnold H, Pette D. Binding of glycolytic enzymes to structure proteins of the muscle. Eur J Biochem. 1968;6:163-171. [Medline] [Order article via Infotrieve]
10.
Lachaal M, Berenski CJ, Kim J, Jung CY. An
ATP-modulated specific association of
glyceraldehyde-3-phosphate dehydrogenase with human
erythrocyte glucose transporter. J Biol
Chem. 1990;265:15449-15454.
11. Dagher SM, Hultin HO. Association of glyceraldehyde-3-phosphate dehydrogenase with the particulate fraction of chicken skeletal muscle. Eur J Biochem. 1975;55:185-192. [Medline] [Order article via Infotrieve]
12. Walsh TP, Masters CJ, Morton DJ, Clarke FM. The reversible binding of glycolytic enzymes in ovine skeletal muscle in response to tetanic stimulation. Biochim Biophys Acta. 1981;675:29-39. [Medline] [Order article via Infotrieve]
13. Walsh TP, Winzor DJ, Clarke FM, Masters CJ, Morton DJ. Binding of aldolase to actin-containing filaments: evidence of interaction with the regulatory proteins of skeletal muscle. Biochem J. 1980;186:89-98. [Medline] [Order article via Infotrieve]
14. Clarke FM, Stephan P, Huxham G, Hamilton D, Morton DJ. Metabolic dependence of glycolytic enzyme binding in rat and sheep heart. Eur J Biochem. 1984;138:643-649. [Medline] [Order article via Infotrieve]
15. Clarke FM, Masters CJ. On the association of glycolytic enzymes with structural proteins of skeletal muscle. Biochim Biophys Acta. 1975;381:37-46. [Medline] [Order article via Infotrieve]
16. Hazen H. The rapid and reversible association of phosphofructokinase with myocardial membranes during myocardial ischemia. FEBS Lett. 1994;339:213-216. [Medline] [Order article via Infotrieve]
17.
Bennardini F, Wrzosek A, Chiesi M.
B-Crystallin in cardiac tissue: association with actin and desmin
filaments. Circ Res. 1992;71:288-294.
18.
Di Lisa F, Menabò R, Barbato R, Siliprandi
N. Contrasting effects of propionate and
propionyl-L-carnitine on energy-linked processes in
ischemic hearts. Am J Physiol. 1994;267:H455-H461.
19.
Molina y Vedia L, McDonald B, Reep B, Brune B, Di
Silvio M, Billiar TR, Lapetina GE. Nitric oxide-induced
S-nitrosylation of glyceraldehyde-3-phosphate
dehydrogenase inhibits enzymatic activity and increases
endogenous ADP-ribosylation. J Biol
Chem. 1992;267:24929-24932.
20. Pagani ED, Solaro RJ. Methods for measuring functional properties of sarcoplasmic reticulum and myofibrils in small samples of myocardium. Methods Pharmacol. 1984;5:49-61.
21. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. [Medline] [Order article via Infotrieve]
22.
Towbin H, Staehelin T, Gordon J. Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proc Natl Acad
Sci U S A. 1979;76:4350-4354.
23.
Saggin L, Ausoni S, Gorza L, Sartore S, Schiaffino
S. Troponin T switching in the developing rat heart.
J Biol Chem. 1988;263:18488-18492.
24.
Saggin L, Gorza L, Ausoni S, Schiaffino S.
Troponin I switching in the developing heart. J
Biol Chem. 1989;264:16299-16302.
25.
Gorza L, Sartore S, Thornell LE, Schiaffino S.
Myosin types and fiber types in cardiac muscle, III: nodal conduction
tissue. J Cell Biol. 1986;102:1758-1766.
26. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685. [Medline] [Order article via Infotrieve]
27. James P, Quadroni M, Carafoli E, Gonnet G. Protein identification by mass profile fingerprinting. Biochem Biophys Res Commun. 1993;195:58-64.[Medline] [Order article via Infotrieve]
28. Wagenknecht B, Freudenrich CC, Lefurgey A, Lieberman M. Calcium depletion and repletion in cultured chick heart-muscle cells. J Mol Cell Cardiol. 1994;26:797-808. [Medline] [Order article via Infotrieve]
29.
Cingolani HE, Koretsune Y, Marban E. Recovery of
contractility and pHi during respiratory
acidosis in ferret hearts: role of Na+-H+
exchange. Am J Physiol. 1990;259:H843-H848.
30. Nayler WG, Ferrari R, Poole Wilson PA, Yepez CE. A protective effect of a mild acidosis on hypoxic heart muscle. J Mol Cell Cardiol. 1979;11:1053-1071.[Medline] [Order article via Infotrieve]
31. Bindoli A, Cavallini L, Rigobello MP, Coassin M, Di Lisa F. Modification of the xanthine-converting enzyme of perfused rat heart during ischemia and oxidative stress. Free Radic Biol Med. 1988;4:163-167. [Medline] [Order article via Infotrieve]
32. Cole HA, Perry SV. The phosphorylation of troponin I from cardiac muscle. Biochem J. 1975;149:525-533. [Medline] [Order article via Infotrieve]
33. Sanders C, Steward DIH, Smillie LB. Troponin-T and glyceraldehyde-3-phosphate dehydrogenase share a common antigenic determinant. J Muscle Cell Motil. 1987;8:118-124.
34.
Spector A, Chiesa R, Sredy J, Garner W.
cAMP-dependent phosphorylation of bovine lens
alpha-crystallin. Proc Natl Acad Sci U S A. 1985;82:4712-4716.
35. Toyo Oka T, Ross J Jr. Ca2+ sensitivity change and troponin loss in cardiac natural actomyosin after coronary occlusion. Am J Physiol. 1981;240:H704-H708.
36. Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987;166:368-379. [Medline] [Order article via Infotrieve]
37. Di Lisa F, De Tullio R, Salamino F, Barbato R, Melloni E, Siliprandi N, Schiaffino S, Pontremoli S. Specific degradation of troponin T and I by m-calpain and its modulation by substrate phosphorylation. Biochem J. 1995;308:57-61.
38. Arnold H, Pette D. Binding of aldolase and triosephosphate dehydrogenase to F-actin and modification of catalytic properties of aldolase. Eur J Biochem. 1970;15:360-366. [Medline] [Order article via Infotrieve]
39. Huitorel P, Pantaloni D. Bundling of microtubules by glyceraldehyde-3-phosphate dehydrogenase and its modulation by ATP. Eur J Biochem. 1985;150:265-269. [Medline] [Order article via Infotrieve]
40.
Horwitz J. -Crystallin can function as a
molecular chaperone. Proc Natl Acad Sci U S A. 1992;89:10449-10453.
41.
Meyer Siegler K, Mauro DJ, Seal G, Wurzer J, deRiel JK,
Sirover MA. A human nuclear uracil DNA glycosylase is the 37-kDa
subunit of glyceraldehyde-3-phosphate
dehydrogenase. Proc Natl Acad Sci U S A. 1991;88:8460-8464.
42. Karpel RL, Merkler DJ, Flowers BK, Delahunty MD. Involvement of basic amino acids in the activity of a nucleic acid helix-destabilizing protein. Biochim Biophys Acta. 1981;654:42-51. [Medline] [Order article via Infotrieve]
43.
Singh R, Green MR. Sequence-specific binding
of transfer RNA by glyceraldehyde-3-phosphate
dehydrogenase. Science. 1993;259:365-368.
44.
Nagy E, Rigby WF.
Glyceraldehyde-3-phosphate dehydrogenase selectively
binds AU-rich RNA in the NAD(+)-binding region (Rossmann fold).
J Biol Chem. 1995;270:2755-2763.
45. Kawamoto RM, Caswell AH. Autophosphorylation of glyceraldehydephosphate dehydrogenase and phosphorylation of protein from skeletal muscle microsomes. Biochemistry. 1986;25:657-661. [Medline] [Order article via Infotrieve]
46.
Robbins AR, Ward RD, Oliver C. A mutation in
glyceraldehyde-3-phosphate dehydrogenase alters
endocytosis in CHO cells. J Cell Biol. 1995;130:1093-1104.
47. Gorza L, Menabò R, Bergamini C, Di Lisa F. Post-ischemic reperfusion is accompanied by changes of troponin T immunoreactivity due to cross-linking. Circulation. In press.
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