© 1999 American Heart Association, Inc.
Original Contribution |
Troponin I Degradation and Covalent Complex Formation Accompanies Myocardial Ischemia/Reperfusion Injury
From the Department of Physiology, Queen's University, Kingston, Ontario, Canada.
Correspondence to Dr J.E. Van Eyk, Department of Physiology, Room 429 Botterell Hall, Queen's University, Kingston, Ontario, Canada K7L 3N6. E-mail jve1{at}post.queensu.ca
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Abstract |
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Abstract—Selective troponin I (TnI) modification has been demonstrated to be in part responsible for the contractile dysfunction observed with myocardial ischemia/reperfusion injury. We have isolated and characterized modified TnI products in isolated rat hearts after 0, 15, or 60 minutes of ischemia followed by 45 minutes of reperfusion using affinity chromatography with cardiac troponin C (TnC) and an anti-TnI antibody, immunological mapping, reversed-phase high-performance liquid chromatography, and mass spectrometry. Rat cardiac TnI becomes progressively degraded from 210 amino acid residues to residues 1–193, 63–193, and 73–193 with increased severity of injury. Degradation is accompanied by formation of covalent complexes between TnI 1–193 and, respectively, TnC residues 1–94 and troponin T (TnT) residues 191–298. The covalent complexes are likely a result of isopeptide bond formation between lysine 193 of TnI and glutamine 191 of TnT by the cross-linking enzyme transglutaminase. With severe ischemia, cellular necrosis results in specific release of TnI 1–193 into the reperfusion effluent and TnT degradation in the myocardium (25-, 27-, and 33-kDa products). Two-dimensional electrophoresis demonstrated that phosphorylation of TnI prevents ischemia-induced degradation. This study characterized the modified TnI products in isolated rat hearts reperfused after a brief or severe period of ischemia, revealing the progressive nature of TnI degradation, changes in phosphorylation, and covalent complexes with ischemia/reperfusion injury. Finally, we propose a model for ischemia/reperfusion injury in which the extent of proteolytic and transglutaminase activities ultimately determines whether apoptosis or necrosis is achieved.
Key Words: protein degradation • myocardial ischemia • myofilament • troponin I • transglutaminase
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Myocardial ischemia/reperfusion presents a spectrum of injury, from reversible damage with mild ischemia, to irreversible cellular necrosis with severe ischemia. Identification of proteins modified during ischemia/reperfusion is fundamental to our understanding of the pathological processes involved. Several laboratories have shown that myofilament proteins, including the troponin complex, are specifically degraded or modified under ischemia/reperfusion.1 2 3 4 5 6 7 8 9 However, the modified troponin products produced by ischemia/reperfusion injury are not yet identified or characterized.
The troponin complex is the regulatory element of the myofilament, which mediates the calcium dependence of muscle contraction in both cardiac and skeletal muscle. Its 3 components, troponin I (TnI), troponin C (TnC), and troponin T (TnT), interact with each other and other thin filament proteins (eg, actin and tropomyosin) through both calcium-dependent and -independent associations.10 11 Calcium binding to TnC at the N-terminal regulatory site produces a conformational change in TnC and movement of TnI away from actin-tropomyosin. Cardiac (c) TnI has a 30 to 32 amino acid N-terminal extension compared with skeletal (s) TnI, which decreases the affinity of TnI for TnC when phosphorylated by protein kinase A (PKA).12 13 The calcium-dependent movement of TnI away from actin reveals a tropomyosin binding site, which results in movement of tropomyosin away from high-affinity myosin binding sites on actin.10 11
Several studies have demonstrated selective TnI degradation1 2 3 4 5 6 7 under ischemia/reperfusion injury, as well as changes in TnT immunoreactivity.8 9 However, each study uses different ischemia/reperfusion periods and different models of ischemic damage (eg, coronary artery occlusion, global ischemia, Langendorff perfusion, and various other combinations).
Myofilament regulatory proteins1 2 3 4 and
structural/cytoskeletal proteins5 6 14 have both been
shown to be susceptible to cleavage or loss under myocardial
ischemia/reperfusion. Mild ischemia/reperfusion has
been shown to disrupt microtubules14 and produces lesions
resulting from loss of desmin, 
-actinin, and
spectrin.15 16 The contractile proteins actin, myosin,
tropomyosin, and TnT are lost from globally ischemic human left
ventricle tissue.6 In particular, TnI has been shown to be
proteolytically cleaved during ischemia and
ischemia/reperfusion.2 3 Different groups have
drawn varying conclusions about the extent and functional importance of
TnI degradation2 3 17 ; however, it is generally agreed
that TnI degradation occurs and correlates to contractile
dysfunction.
In this study, we have isolated and characterized the modifications to TnI in both mildly and severely ischemic isolated perfused rat hearts. Through the use of tricine-SDS-PAGE (T-PAGE) and Western blot analysis, we have shown that TnI degradation is a progressive process. Increases in the severity of the ischemic insult lead to more extensive degradation of TnI and preferential release of a TnI degradation product into the reperfusion effluent. Strong indirect evidence has shown that the calcium-dependent protease calpain is likely responsible for the degradation of TnI.2 18 We have also found covalent complexes of TnI/TnC and TnI/TnT that appear with mild ischemia but disappear with more severe injury. The cross-linking enzyme transglutaminase (TGase) is proposed to be responsible for the formation of covalent bonds between the troponin subunits.8 These modifications within the troponin complex may be important in the global dysfunction of the heart, because of disruption of the calcium regulation of contraction. This study is the first to identify the TnI degradation products and covalent complexes produced by myocardial ischemia/reperfusion injury. Various parts of our results have been reported in abstract form.19
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Materials and Methods |
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Langendorff Perfusion of Isolated Rat Hearts
We adapted this technique from a previously published protocol.1 Animals were used in compliance with the Animals for Research Act (Province of Ontario) and the Canadian Council on Animal Care. Male Sprague-Dawley rats (250 to 275 g) were anesthetized with an overdose of sodium pentobarbital (>100 mg/kg) and heparinized via the inferior vena cava (50 mg/kg). Hearts were quickly excised and placed in ice-cold saline. The aorta was cannulated on a 1.6-mm glass cannula (Radnoti Glass Inc) and perfused in a nonrecirculating apparatus with heart chamber (Radnoti Glass Inc) at 14 mL/min with Krebs-Ringer bicarbonate buffer ([in mmol/L] sodium chloride 100, potassium chloride 4.74, potassium dihydrogen phosphate 1.18, magnesium sulfate 1.18, calcium chloride 1.15, sodium dihydrogen carbonate 25, glucose 11.5, sodium pyruvate 4.92, and sodium fumarate 5.39, pH 7.4) equilibrated with 95% O2/5% CO2 at 37°C. The right atrium was fitted with electrodes, and the hearts were paced at 360 bpm. Hearts were subjected to 15 minutes of equilibration followed by 0, 15, or 60 minutes of ischemia (no flow and no stimulation) and 45 minutes of reperfusion (0/45, 15/45, and 60/45, respectively). The effluent from the initial 10 minutes of reperfusion was collected, lyophilized, and stored at –20°C until analysis. We have previously shown that the majority of proteins are released in the first 10 minutes of reperfusion.1 The left ventricle was immediately frozen in liquid nitrogen and stored at –70°C until analysis.
Isolation and Identification of Modified TnI Products
Tissue Homogenization
Left ventricles (n=3) were homogenized together in
20 mmol/L Tris-HCl, pH 7.4, 6 mol/L urea, and 200 mmol/L
potassium chloride, with a protease inhibitor cocktail
(3.6 µmol/L leupeptin, 2.1 µmol/L pepstatin A, and
50 µmol/L phenylmethylsulfonylfluoride) on ice. Aliquots
of tissue homogenates for electrophoresis were stored at
–20°C until use. Remaining tissue homogenates for
affinity chromatography were dialyzed at 4°C against
2 mol/L urea, 1 mol/L potassium chloride, 1 mmol/L DTT, and
20 mmol/L Tris-HCl, pH 7.4, with the protease
inhibitor cocktail, followed by sequential dialysis against
1 mmol/L DTT, 0.1 mmol/L EDTA, 20 mmol/L Tris-HCl, pH 7.4,
and protease inhibitor cocktail, with decreasing
concentration of potassium chloride from 1 mol/L to 500 and 200
mmol/L (2 changes). In the final 2 dialysis steps (200 mmol/L
potassium chloride), DTT was not present, and tissue samples were
maintained in reduced form with nitrogen gas bubbled through the
dialysis buffer. Samples were centrifuged at 15 000 rpm for 10
minutes at 4°C, and the supernatant was used for further
analysis. Total protein concentration of tissue
homogenate supernatants and reperfusion effluents were
determined according to Lowry et al.20 Bovine cTnI, cTnC,
and cTnT and rabbit sTnC were isolated and purified as published by
Ingraham and Hodges.21
Affinity Chromatography
Rabbit sTnC was cross-linked to 3M Emphaze resin (Pierce)
according to the manufacturer's protocol. The anti-TnI monoclonal
antibody (MAb) 8I-7 (Spectral Diagnostics, Figure 1
) was cross-linked to cyanogen bromide
Sepharose (Pharmacia) according to the manufacturer's protocol. Both
affinity columns were equilibrated in buffer A consisting of 20
mmol/L Tris-HCl, pH 7.4, 50 mmol/L potassium chloride, and 1
mmol/L calcium chloride with the protease inhibitor
cocktail. Left ventricle tissue supernatants were loaded onto either
column (
10 mg), and the column was washed with 10 volumes of buffer
A. Bound proteins were eluted with 65 mmol/L glycine-HCl, pH 3.2.
Fractions (1 mL) were collected into tubes containing 100 µL of 0.86
mol/L MOPS, pH 8.0, to neutralize the pH to 7.4. After lyophilization,
fractions were resuspended in 0.05% aqueous trifluoroacetic acid and
analyzed by reversed-phase high-performance liquid
chromatography (RP-HPLC), using an analytical Zorbax C8
300SB RP column (4.6 mm internal diameterx250 mm,
Chromatographic Specialists Inc). The HPLC system consisted
of a Varian (Missisauga, Canada) 9100 autosampler, 9012 solvent
delivery system, and 9065 diode ray detector. The proteins were
eluted using an AB solvent system, in which solvent A was composed of
0.05% aqueous trifluoroacetic acid and solvent B was composed of
0.05% trifluoroacetic acid in acetonitrile. The AB gradient consisted
of an isocratic hold (100% solvent A) for 5 minutes followed by a 2%
solvent B/min linear gradient at a flow rate of 1 mL/min. The peaks
were collected, lyophilized, and analyzed by mass spectrometry,
Western blotting, and amino acid microsequencing (Alberta Peptide
Institute, University of Alberta, Edmonton, Canada).
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Gel Electrophoresis and Western Blotting
Tissue homogenates, effluent samples, and the peaks
collected from RP-HPLC analysis of anti-TnI MAb affinity
chromatography-bound fractions were separated by 12.5%
SDS-PAGE, or by T-PAGE22 in the presence of 6 mol/L urea,
using the Mini-gel system (Bio-Rad). T-PAGE was performed with a 10%T
(total acrylamide concentration), 3%C (concentration of
bis-acrylamide) resolving gel and 4%T, 3%C stacking gel
containing 6 mol/L urea, 0.1% SDS, and 0.1 mol/L Tris-HCl, pH 8.45.
The cathode running buffer consisted of 0.1 mol/L Tris-HCl, pH 8.25,
0.1 mol/L tricine, and 0.1% SDS, and the anode buffer consisted of 0.2
mol/L Tris-HCl, pH 8.9. Samples were diluted 2-fold with 2% SDS,
5 mmol/L Tris-HCl, pH 6.5, 20% sucrose, 0.05% bromophenol blue,
and 100 mmol/L ß-mercaptoethanol (ß-ME), with the addition of
6 mol/L urea for T-PAGE. Prepared samples were boiled for 5 minutes and
loaded onto the gel. Gels were stained with Coomassie blue followed by
silver23 or transferred to a nitrocellulose (for Western
blotting) or polyvinylidine difluoride (for amino acid
microsequencing) membrane using a wet transfer apparatus
(Bio-Rad) with 10 mmol/L 3-cyclohexylamino-1-propanesulfonic acid
(CAPS), pH 11.0, for 16 hours at 4°C. Western blot analysis
was carried out as described previously.1 The primary
antibodies (Figure 1
) were detected with goat anti-mouse IgG
conjugated to alkaline phosphatase (Jackson Immuno Research
Laboratories) and CDP-Star chemiluminescence reagent (NEN-Mandel).
Epitope mapping of the MAbs 8I-7 and 3I-35 (both Spectral
Diagnostics, Toronto, Canada) was performed by
Western blotting of 12.5% SDS-PAGE–separated recombinant rat cardiac
(rc) TnI fragments, synthetic peptide, or BSA-conjugated rcTnI
peptide (Figure 1), as described elsewhere.1 The
MAb AM-IN was obtained from, and the epitope mapped by, Dr J. Ladenson
(Washington University, St. Louis, Mo) and binds the N-terminal region
of TnI. MAb TnT clone JLT-12 was obtained from Sigma, and the epitope
is not known. All TnT Western blots were confirmed with a second
anti-TnT MAb (Spectral Diagnostics, Toronto, Canada), also of unknown
epitope. The epitope of MAb 3I-59 on rcTnI is not known, although it
binds preferentially to TnI-containing covalent complexes. The MAb 8I-7
Western blots were quantified using Corel Photohouse (version
8).24 The amount of protein loaded onto the gel was
increased from 5 µg (Figure 2B) to 20
µg (Figure 2A) or 40 µg (MAb TnT overexposed/overloaded,
Figure 2A), and overexposure of blots was used to facilitate
visualization of weak associations between the MAbs and the TnI
products.
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Alkaline urea PAGE was performed as outlined by Head and Perry.25 Lyophilized peaks from the RP-HPLC analysis of anti-TnI MAb affinity chromatography-bound fractions were resuspended in buffer containing 6 mol/L urea, 100 mmol/L ß-ME, 20 mmol/L Tris-HCl, pH 8.9, 3 mmol/L calcium chloride, and 0.05% bromophenol blue and loaded onto the gel. The gel consisted of 8%T, 0.8%C resolving and 5%T, 0.8%C stacking gels, with 6 mol/L urea, 20 mmol/L Tris-HCl, and 124 mmol/L glycine at pH 8.6. The gel was stained with Coomassie blue and silver.23
Two-dimensional (2D) electrophoresis of tissue homogenates was adapted from a standard protocol (Bulletin 1144) from Bio-Rad, except that we used T-PAGE for the second dimension. Proteins were resolved in the first dimension by isoelectric focusing on a mini-Protean II isoelectric focusing gel electrophoresis apparatus (Bio-Rad) using an ampholyte mixture of 90% pH 3.5 to 10.0 (Sigma) and 10% pH 4.0 to 6.5 (Pharmacia). Protein resolution in the second dimension was carried out by T-PAGE with 6 mol/L urea. The 2D gels were stained with Coomassie blue or transferred to nitrocellulose for Western blot analysis.
Mass Spectrometry
Approximately 50 to 200 µg of each lyophilized RP-HPLC peak
obtained from both affinity columns was analyzed by
matrix-assisted laser desorption/ionization (Kratos) or electrospray
(Fisons VG Quattro) mass spectrometry. From these masses, the possible
TnI, TnC, and TnT fragments were determined using the SwissProt protein
database and the PeptideMass tool from the WorldWide Web molecular
biology server of the Swiss Institute of Bioinformatics
(http://expasy.hcuge.ch/). Protein sequences from rcTnI, mouse cTnC
(mcTnC), and rcTnT were sequentially clipped from the amino and
carboxyl termini until a match to the observed monoisotopic mass was
found. Masses and amino acid sequences of the TnC and TnT components of
the high molecular weight complexes were determined by considering all
amino acid sequences that conformed to the necessary restrictions, as
described in Results.
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Results |
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Time Dependence of TnI Modification
In this study we have identified the TnI degradation products and characterized additional modifications within the troponin complex that are induced by varying degrees of ischemia/reperfusion injury. Figure 2A
shows Western blots of T-PAGE–separated (with
6 mol/L urea) left ventricular tissue from isolated
Langendorff-perfused rat hearts that had undergone 0, 15, or 60 minutes
of no-flow ischemia followed by 45 minutes of reperfusion. With
this gel system, the visualization of 6- to 70-kDa proteins is
facilitated, but some proteins, including the troponin subunits, do not
migrate according to their molecular weights. Initial Western blot
analysis of modified TnI products was performed using the
following series of MAbs that recognize various defined regions of TnI:
AM-IN (N-terminal), 8I-7 (central), and 3I-35 (C-terminal) (Figure 1). Control hearts (0/45) showed little degradation of TnI
(5.6%) (Figure 2A, Table 1A). A
substantial increase in the quantity of degradation was shown under
ischemia/reperfusion, with increases in the amount and type of
TnI products corresponding to increases in the severity of the
ischemic insult. The mildly ischemic condition of
15/45, in which few or no proteins are released indicating no cellular
necrosis,1 resulted in modification of 48% of total TnI,
because of production of a 22-kDa TnI degradation product
and 3 high molecular mass complexes (55, 66, and 75 kDa;
combined, 16.9% of total TnI) (Figure 2A, Table 1A).
Analysis by 12.5% SDS-PAGE demonstrated that these high
molecular mass complexes are all in fact between 30 and 40 kDa (Figure 2B). The proportion of the 22-kDa product to total TnI
content did not change substantially in tissue from 60/45 hearts
(21.3%, Table 1). This is in spite of an increase in the
quantity of TnI degradation (64.7%, Table 1) due to changes in
the type of TnI modification. Severe ischemia/reperfusion
injury resulted in loss of the high molecular weight complexes, with
further degradation of TnI to 2 products with molecular masses of
16 and 15 kDa (15.1% and 17.2% of total TnI, respectively; Table 1), as well as degradation of TnT (Figure 2A). The
quantification of MAb 8I-7 Western blots was confirmed with anti-TnI
MAb E2 (epitope rcTnI residues 136–147), with all modified TnI
products within a 5% error of those obtained with MAb 8I-7 (data
not shown). We recognize, however, that the inherent assumption of
equal binding affinity of these MAbs for all products does not
necessarily hold. Significant cellular necrosis occurs with
60/45,1 and analysis of 60/45 reperfusion effluent
demonstrated preferential release of the 22-kDa TnI product from
the cell with respect to intact TnI (Figure 2B). Previously
published analysis has demonstrated that no proteins are
released into the reperfusion effluent with 15/45, and so no data are
included.1 Analysis of 60/45 reperfusion effluent
with MAb 3I-59, which preferentially binds to the covalent complexes,
demonstrated that TnI-containing covalent complexes are also found in
the effluent (Figure 2B). Previously we used a different
anti-TnI antibody (MAb E2) which failed to demonstrate the presence of
TnI products in the reperfusion effluent.1 This is
similar to the results shown in Figure 2B for MAb 3I-35. Both
MAbs E2 and 3I-35 only weakly associate with the 22-kDa degradation
product. MAbs 8I-7 and AM-IN clearly detect the 22-kDa degradation
product, strongly indicating that this product is indeed
derived from TnI.
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The ability of the anti-TnI MAbs to recognize different epitopes on TnI
was exploited in the initial characterization of the modified TnI
products observed in ischemia/reperfused
myocardium. We have previously shown that the 22-kDa TnI
degradation product is a product of C-terminal proteolysis
because of its weak interaction with MAb 10F2 (epitope rcTnI amino acid
residues 188–199).1 This is confirmed here by its weak
associations with MAb 3I-35, which has an epitope similar to that of
10F2 on TnI (Figure 1). The 16- and 15-kDa products that
appear with severe ischemia are degraded from both the N- and
C-termini as shown by their reduced affinity for both 3I-35 and AM-IN
MAbs. Taken together, these results suggest that
ischemia/reperfusion-induced TnI modification results in an
initial C-terminal cleavage, followed by progressive N-terminal
degradation with increases in the severity of the ischemic
insult.
The high molecular weight complexes seen in 15/45 tissue were not
disrupted by 6 mol/L urea, 0.1% SDS, and 100 mmol/L ß-ME,
indicating that a TnI product(s) was covalently linked in a
nondisulfide bond to some other protein. The 55- and 66-kDa covalent
complexes interacted strongly with 8I-7 and AM-IN MAbs but only weakly
with MAb 3I-35, indicating C-terminal degradation, possibly similar to
that of the 22-kDa degradation product (compare response to 8I-7
versus 3I-35, Figure 2A). The 66- and 75-kDa covalent complexes
interacted strongly with the anti-TnT MAb. The 3 complexes may be
degradation products of TnI/TnC (55 kDa), TnI/TnT (66 kDa), and
TnT/TnC (75 kDa) (Table 2).
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Western blot analysis of 2D electrophoresis of 60/45 tissue
homogenate revealed at least 2 spots, or
phosphorylation states, for intact TnI, with only 1
spot for each of the degradation products (Figure 2C). Since
phosphorylation adds negative charge to proteins,
phosphoproteins appear as spots at the same molecular weight as when
unphosphorylated, but at lower pH. The degradation
products are thus not phosphorylated.
Isolation and Identification of Modified TnI Products
Affinity chromatography was used to isolate the
various modified TnI products that were observed by Western
blotting of the ischemia/reperfused tissue (Figure 2).
TnC affinity chromatography of the 60/45 tissue sample
resulted in the elution of a single TnI product by RP-HPLC (Figure 3A). This product was collected and
analyzed by mass spectrometry, which detected a mass of
22 144±7.8 Da (Figure 3B). The identity of this product as
the 22-kDa TnI degradation product was confirmed by Western
blotting (data not shown). Analysis of the rcTnI amino acid
sequence identified a single sequence, rcTnI residues 1–193 (mass
22 152 Da), which was the only sequence that corresponded to the
observed mass. The identification of TnI 1–193, a C-terminal
degradation product of rcTnI, confirmed the weak interaction with
the C-terminal MAb 3I-35 (Figure 2A) and the previously reported
blocked N-terminus as revealed by unsuccessful amino acid
microsequencing.1
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Although TnI 1–193 is the primary TnI degradation product observed
with 15/45, more severe ischemia results in further degradation
of TnI (Figure 2A). These other products were isolated by
anti-TnI MAb 8I-7 affinity chromatography of 15/45 and
60/45 left ventricle tissue. Several protein products were isolated
from fractions that bound to the MAb 8I-7 affinity column by RP-HPLC
(Figure 4A). While 15/45 tissue did not
yield sufficient protein for further analysis, 60/45 tissue
produced 4 distinct peak groups (referred to as peaks 1 through 4,
Figure 4A) that were further analyzed by mass
spectrometry (Table 2), T-PAGE with 6 mol/L urea (Figure 4B), Western blotting (Figure 4C), and alkaline urea PAGE
(Figure 4D).
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Peak 1 comprised a series of degradation products (molecular
masses 25, 27, and 33 kDa, Figure 4B), and Western blotting
(Figure 4C) confirmed their identity as the TnT degradation
products observed previously (Figure 2A). Peak 2 comprised a
high molecular weight covalent complex, in addition to several smaller
degradation products. The covalent complex was shown to comprise
TnI (MAb 8I-7 overexposure, Figure 4C) and TnC (alkaline urea
PAGE, Figure 4D). The other components of peak 2 were shown to
be intact rcTnI, the 22-kDa product (TnI 1–193), and the 16-
and 15-kDa products (MAb 8I-7 overexposure, Figure 4C). Mass
spectrometry of this peak returned masses of 32 734±14 Da,
15 348±15 Da, and 14 096 Da (Table 2). Analysis of
the rcTnI amino acid sequence identified single sequences corresponding
to the 2 smaller masses: rcTnI 63–193 (mass 15 377 Da) and rcTnI
73–193 (mass 14 096 Da). Peak 3 comprised a large number of high
molecular weight products. Western blots demonstrated the presence
of 1 TnI-containing covalent complex and intact rcTnI, with little
further degradation (MAb 8I-7 overexposure, Figure 4C). The
covalent complex in peak 3 did not contain TnC (Figure 4D) but
did contain TnT (MAb TnT overexposed/overloaded, Figure 4C).
Mass spectrometry of peak 3 returned a mass of 32 872±9 Da for the
complex. Peak 4 comprised a covalent complex and a protein with
electrophoretic migration and staining properties similar to those of
cTnC (Figure 4B). TnT was shown to be a component of the
covalent complex (MAb TnT overexposed/overloaded, Figure 4C),
while alkaline urea PAGE confirmed that both of these products
contained TnC. Importantly, TnI was not present in this covalent
complex (MAb 8I-7, Figure 4C and 4D). Mass spectrometry of peak
4 returned a mass of 33 595±32 Da for the complex and a mass of
18 420 Da for the smaller product. The 18 420-Da product is
similar in mass to mcTnC, but since the sequence for rcTnC is not
known, further analysis was precluded. This analysis is
summarized and correlated to the Western blotting data in Table 2.
The cumulative information about the covalent complexes from
Western blotting, mass spectrometry, and alkaline urea PAGE, as
summarized in Table 2, was used to assign identities to the 2
TnI-containing covalent complexes. There was insufficient information
about the 33 595-Da TnT-TnC covalent complex to perform similar
analyses for it, and so it is not included in Table 2.
The 2 TnI-containing complexes have identical immunoreactivities to
that of the TnI MAbs (Figures 2A and 4C), which
correspond to that of TnI 1–193 (Figure 2A), not intact TnI
(Figure 2A). Few processes are known to cause covalent
cross-linking, except through the enzyme TGase, which is known to
target the troponins.26 TGase is a ubiquitous enzyme that
forms isopeptide bonds between specific glutamine and lysine
residues,26 27 and the C-terminal residue of rcTnI 1–193
is a lysine. Amino acid microsequencing of these complexes was
unsuccessful, likely because of a blocked N-terminus. Even so, the
amino acid sequences of mcTnC and rcTnT were analyzed to
determine whether there were sequences that would sum with rcTnI 1–193
to the appropriate mass and have either a blocked N-terminus or an
N-terminal glutamine residue. While isopeptide bonds are still
susceptible to hydrolysis, the sequence produced by linking TnI lysine
193 to the N-terminus of another protein or degradation product
would be linear and not susceptible to Edman degradation. This led to
the identification of single sequences, mcTnC 1–94 (theoretical mass
of complex with TnI 1–193 of 32 730 Da) and rcTnT 191–298
(theoretical mass of complex with TnI 1–193 of 32 871 Da), with these
properties (Table 2). The N-terminus of rcTnC is known to be
blocked, and rcTnT residue 191 is indeed a glutamine.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TnI Degradation Is a Progressive and Selective Process
Several groups have examined the cellular modifications associated with myocardial ischemia/reperfusion injury, including the degradation of myofilament proteins such as TnI and
-actinin.
Westfall and Solaro4 first postulated degradation of TnI
and TnT following 60 minutes of complete global ischemia in rat
hearts. Gao et al2 detected the presence of a TnI
degradation product in mildly ischemic (20-minute
ischemia and 20-minute reperfusion) isolated perfused rat
hearts, and this injury was prevented by low calcium/low pH
perfusion. Hein et al6 demonstrated changes in the
localization pattern of myosin, actin, tropomyosin, and TnT as early as
10 minutes after the onset of ischemia in
cardiomyopathic human left ventricular
tissue. Recently, our laboratory demonstrated that TnI and -actinin
in isolated rat hearts are specifically degraded during 15/45, 60
minutes of ischemia with no reperfusion, and
60/45.1 Here we have identified and characterized the
modifications to TnI under mild and severe ischemia/reperfusion
and demonstrated a progressive and selective pattern to the
modification process, whereby increases in the severity of
ischemia result in increases in the extent of TnI
degradation.
The mild ischemia/reperfusion condition of 15/45 results in the
activation of 2 processes through increases in intracellular
calcium,2 which are protease activity and covalent
cross-linking activity. There is substantial indirect evidence
suggesting that the calcium-dependent cysteine protease calpain is
responsible for the production of the 22-kDa TnI C-terminal
degradation product, identified here as rcTnI 1–193 (Figure 5, Table 2). Calpain may also be
implicated in the N-terminal proteolytic activity, which produces
further degradation of TnI to residues 63–193 and 73–193, with more
severe ischemia/reperfusion (60/45). Calpain is known to cleave
both rcTnI and rcTnT in vitro,18 and treatment of skinned
trabecula fibers with calpain produces a dysfunction and
TnI degradation product similar to those seen in
ischemia/reperfused fibers.7 To date, there is no
known consensus sequence specifically targeted by calpain, and the 3
rcTnI cleavage sites identified here share no significant sequence
similarity. Currently, there is a dispute over whether sequences rich
in proline, glutamic acid, serine, and tyrosine residues ("PEST"
sequences) are involved in recruitment of calpain to cleavage
sites.28 29 30 The processes by which the activated
enzyme is localized to the thin filament are only now being
postulated.
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The functional implications of TnI degradation are more readily
interpreted now that the amino acid sequence of the degradation
products is known. Studies of rcTnI deletion mutants comprising
amino acid residues 1–199, 1–188, and 1–151 have shown that the
1–188 and 1–151 mutants have impaired abilities to bind TnC and
inhibit actin-activated myosin ATP hydrolysis compared with
both the 1–199 mutant and intact rcTnI.31 Neighboring
residues between rcTnI 150–180 have been implicated in both TnC and
actin-tropomyosin binding.32 The decrease in calcium
sensitivity or in maximum force seen in triton-skinned
trabecula fibers from ischemia/reperfused rat
hearts cannot yet be attributed to rcTnI 1–193, the primary
ischemia-induced degradation product.1 7
However, substitution of the troponin complex from stunned
myocardium into control skeletal muscle fibers demonstrated
a decrease in the calcium sensitivity of force generation with respect
to controls.33 Modification to the troponin complex may be
directly responsible for the myofibrillar dysfunction observed with
ischemia. While substantial intact TnI exists within the
ischemic cells (Table 1A), several groups have found
that even low levels of modified contractile proteins can significantly
alter contractile function.34 35 To further explore the
functional implications of TnI degradation, transgenic mice have been
produced that express mcTnI 1–193 in the myocardium (Dr A.
Murphy, Johns Hopkins University, personal communication, 1998).
These mice demonstrate depressed left ventricular
function.
In contrast, the functional characteristics of the rcTnI N-terminus are well defined, with essential roles in TnC binding and the cooperativity of actin-tropomyosin binding.10 32 The progressive loss of this region with severe ischemia may result in a reduction in the ability of calcium to regulate the contraction of cardiac muscle.
Previous work has shown that 15/45 conditions do not result in cellular necrosis over control levels, while 60/45 conditions do result in necrotic release of cellular proteins.1 These experimental conditions approximate the clinical phenomena of myocardial stunning and acute myocardial infarction. The rcTnI component of these proteins was shown here to be primarily rcTnI 1–193 and not intact rcTnI. Also present in the reperfusion effluent were TnI-containing covalent complexes. It has been established that cTnI products are found in the blood after an ischemic episode, and serum TnI levels are now used as a clinical diagnostic for acute myocardial infarction.36 Recent work by Wu et al37 has demonstrated the presence of a binary complex of cTnI-cTnC, a ternary complex of cTnT-cTnI-cTnC, free cTnT, and no free cTnI in serum from patients who had experienced acute myocardial infarction. Our results, along with those of Wu et al37 and others (see references in Wu et al37 ), suggest that current cTnI serum diagnostics may predominantly detect modified TnI products (ie, TnI 1–193 and covalent complexes) and little if any intact protein.
While modified TnI products were produced with both mild and severe
ischemia/reperfusion, a population of TnI was protected from
degradation (Figure 4C, compare peaks 2 and 3, MAb 8I-7).
Quantification and comparison of peaks 2 and 3 obtained from RP-HPLC
analysis of bound fractions from 8I-7 affinity
chromatography of 60/45 tissue demonstrated that while
29% of TnI was degraded in peak 2, only 9% was degraded in peak 3
(Table 1B). In vitro studies have shown that
phosphorylation of cTnI by PKA reduces the sensitivity
of cTnI to degradation by calpain.18 Two-dimensional
electrophoresis is a powerful tool for elucidating
phosphorylation states and determined that the TnI
degradation products were indeed unphosphorylated,
despite significant levels of phosphorylated intact TnI
(Figure 2C). However, the 15- and 16-kDa TnI products have
lost the N-terminal phosphorylation sites at serine
residues 23 and 24. Recent work has shown that
phosphorylation of TnI and TnT is increased following
myocardial infarction in dogs,38 confirming that the
amount of phosphorylated TnI seen in the 2D gel is not
excessive. Interestingly, the calpain-mediated proteolysis of other
proteins, in particular tau, is inhibited by substrate
phosphorylation.39 40 In the case of
connexin-32, phosphorylation prevents calpain-mediated
proteolysis but not degradation by papain, -chymotrypsin, proteinase
K, or trypsin.41 Phosphorylation of TnI by
PKA produces a change in the fluorescence properties of
tryptophan 192 of cTnI, indicating that the conformation of the
C-terminal region of cTnI is altered.42 In our laboratory,
we have found that treatment of experimental animals with
isoproterenol, which activates PKA-dependent pathways, provides
protection against TnI degradation (Taylor and Van Eyk, unpublished
data, 1998). The "protected" population of TnI observed in
Figure 4C may represent phosphorylated
rcTnI.
Covalent Complexes Are Formed With Mild
Ischemia/Reperfusion
The identification of binary covalent complexes formed under mild
ischemia/reperfusion injury, comprising TnI 1–193 and,
respectively, TnC 1–94 and TnT 191–298, suggests the activity of a
cardiac tissue TGase isoform. TGases are activated by calcium
to catalyze the formation of isopeptide bonds between specific
glutamine residues and primary amines, preferentially the amine group
of adjacent lysine residues.43 Nakaoka et
al44 demonstrated that -adrenergic receptors of the
heart activate a phospholipase C enzyme through a GTP binding
protein, Gh, which they further demonstrated to
exhibit TGase activity. It has since been shown that all tissue TGases
contain GTP binding domains,27 45 46 and the TGase
activity of Gh is inhibited by the binding of
GTP.27 45 46 Furthermore, Gh
expression is downregulated in the failing human heart, despite an
increase in -adrenergic receptor expression; however, there was no
correlation between TGase activity and Gh protein
levels.47 Hwang et al47 suggest that
Gh activation may be regulated by an unknown
inhibitory factor. However, Gh may
not be the only TGase in cardiomyocytes, and so the exact
ischemia/reperfusion-induced mechanism of TGase activation
remains unclear.
The TnI/TnT complex may be the result of a covalent bond between TnI
lysine 193 and TnT glutamine 191, while the TnI/TnC complex may be the
result of a covalent bond between TnI lysine 193 and an internal
glutamine residue in the TnC 1–94 sequence (Figure 5). Despite
the absence of a sequence for rcTnC, there is a high degree of homology
within cTnC isoforms at both the amino acid and structural levels
between mammalian species. The low-affinity regulatory calcium binding
site of the TnC N-terminus is known to interact with the C-terminal
region of cTnI,10 such that TnI lysine 193 is likely
linked to an N-terminal glutamine on TnC 1–94. The C-terminal region
of cTnC (residues 126–136), which is absent in the
ischemia-induced TnI/TnC complex, is believed to contain an
anchoring region for rcTnI that facilitates the movement of the
inhibitory region (rcTnI residues 137–148) between actin
and TnC.10 32 The covalent complex preserves the
regulatory calcium binding site of TnC, anchors rcTnI to cTnC, and may
modulate the affinity of the inhibitory region of rcTnI for
cTnC.
The covalent complexes observed in myocardium with mild
ischemia/reperfusion were greatly reduced with more severe
conditions, implying that the TGase-catalyzed isopeptide bonds may have
been broken. Although a portion of the covalent complexes were released
into the 60/45 reperfusion effluent (Figure 2A and 2B, Table 1A), this cannot account for all of the complexes observed in
15/45 tissue (Figure 2A). Since the isopeptide bond is not
susceptible to protease activity, the most probable mechanism whereby
these links could be broken is through isopeptidase activity, whereby
the isopeptide bond is hydrolyzed.48 While the presence of
a tissue isopeptidase is currently under dispute,49 50 51
TGases themselves show significant internal isopeptidase activity at
high concentrations.52 It is possible that escalating
TGase activation following longer ischemic episodes results in
a reversal of the covalent linkages formed under milder conditions. The
progress of TGase-induced ischemia/reperfusion injury may
therefore be self-limiting.
The involvement of TGases in ischemia/reperfusion injury has been suggested previously by Gorza et al,8 who observed unidentified covalent complexes containing TnT in ischemia/reperfused cardiomyocytes accompanied by changes in TnT immunoreactivity in cryosections of ischemic rat hearts with 60 minutes of ischemia followed by 30 minutes of reperfusion. However, Gao et al2 did not see covalent complexes with 20 minutes of ischemia followed by 20 minutes of reperfusion. This may reflect the affinity of the MAbs used in analysis of the degradation products and different periods of reperfusion. The lack of a clear activating signal for TGase activity prevents any further conclusions. However, the significant TnT degradation observed here with severe ischemia/reperfusion supports the modulation of TnT immunoreactivity observed by Gorza et al8 and the production of the covalently complexed TnT 191–298. This degradation results in loss of the majority of the rcTnI binding region on cTnT (residues 152–209)53 and loss of N-terminal tropomyosin binding sites.54 55 The formation of a covalent complex may help to preserve the association of the troponins.
Possible Interactions Between Calpain and TGase
The relationship between proteolytic and covalently linking
processes may reflect the balance of the regulatory processes that
ultimately govern cell death. The release of cellular proteins into the
reperfusion effluent with severe ischemia implies significant
necrosis, the acute process whereby the cell membrane of severely
damaged cells ruptures. However, DNA fragmentation studies have
demonstrated that a portion of cardiomyocytes are destroyed
through the highly regulated process of apoptosis, whereby
specific stimuli result in the transcription of cell death genes, and
the cell is dismantled into discrete "packages" (ie, proteins are
not released).56 57 58 Hypoxic damage in the rat liver is
prevented by in vivo transfection with Bcl-2, a known apoptosis
inhibitor.59 Bcl-2 treatment also prevents
potassium cyanide–induced necrotic death in PC 12 cells,
suggesting a link between apoptosis and
necrosis.60 Tissue TGases have been implicated in the
cellular remodeling associated with apoptosis,61
and TnT cross-linking has been found in human apoptotic
cardiomyocytes.8 9 Calpain is known to be
activated under both apoptotic and nonapoptotic
conditions.28 62 Interactions between TGases and calpain
have been previously suggested, since factor XIII (a blood clotting
factor and a TGase) is proteolytically
activated.63 However, the tissue-type TGases lack
the N-terminal extension that is cleaved from factor XIII. Even so,
tissue TGases have been shown to be in situ substrates for calpain
degradation, which is inhibited in the presence of GTP but
inactivates the enzyme.46 The pathological
interactions between TGases and calpain are not yet known. Since the
loss of covalent complexes with severe ischemia is associated
with the further degradation of TnI and TnT, these covalent attachments
may provide some protection against further degradation. This possible
protective function indicates that the 2 processes are associated but
not complementary (ie, destruction versus preservation). The balance
between these 2 processes within the cell may determine whether
necrosis or apoptosis is achieved with increasing muscle
damage.
Proposed Model for Ischemia/Reperfusion-Induced TnI
Modification
The onset of ischemia/reperfusion injury involves an
increase in cellular calcium concentration.1 7 It is this
increase in cellular calcium that may result in the activation of both
proteolytic and TGase activities, possibly through interactions between
calpain and a tissue TGase. The selective degradation of troponin (to
TnI 1–193, cTnC to TnC 1–94, and cTnT to 191–298) may occur either
before or after the formation of covalent links between these
components. The balance between the formation of the stabilized
covalent complex and the amount of troponin degradation may determine
whether necrotic or apoptotic pathways will be
activated. While stunning conditions have not been shown to
produce apoptotic death, pathways that may eventually lead to
apoptosis, as well as those which may lead to necrosis, may be
activated. As a result, TGase and calpain activities are
modulated such that covalent complexes are maintained, broken, or
released into the reperfusion effluent, depending on the severity of
the ischemic insult. The factors that modulate the activity of
these enzymes may include pH, intracellular calcium, and ATP levels, as
well as many others.64 With severe
ischemia/reperfusion, TnI 1–193 released from broken covalent
complexes is further selectively degraded from the N-terminus, to TnI
63–193 and TnI 73–193, producing a progression of degradation
products. As cellular damage proliferates, necrosis ensues. In
cells with only moderate TGase activation, or moderate protease
activation, under more severe ischemia/reperfusion, necrosis
averted through the stabilizing actions of TGase cross-links. This
population of cells is given the opportunity to gain the possible
advantages of undergoing apoptosis instead of necrosis. This
model thus proposes that there are 2 populations of
cardiomyocytes, necrotic and apoptotic, which may
represent histological and/or functional
differences. The spectrum of damage caused by myocardial
ischemia/reperfusion injury is the
physiological manifestation of the protein
modifications induced by the ischemia/reperfusion insult and
the balance between necrosis and programmed cell death processes. The
result is the selective and progressive degradation of cTnI, which is
accompanied by the formation of covalent complexes between the troponin
subunits.
|
Acknowledgments |
|---|
This research was supported by the Medical Research Council of Canada (grant-in-aid No. MT-14375) and the Heart and Stroke Foundation of Canada (grant-in-aid No. T-3759). The authors thank Dr John Strauss, Division of Pre-Clinical Sciences, New York College of Osteopathic Medicine of the New York Institute of Technology, for his assistance and advice in the writing of the manuscript.
Received July 29, 1998; accepted October 21, 1998.
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References |
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B. J. Biesiadecki and R. J. Solaro When Hearts Fail So Does Skeletal Muscle: Breaking a Vicious Cycle Circ. Res., June 23, 2006; 98(12): 1456 - 1458. [Full Text] [PDF]
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W. D. Gao, T. Dai, and D. Nyhan Increased cross-bridge cycling rate in stunned myocardium Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H886 - H893. [Abstract] [Full Text] [PDF]
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G. Sawicki, H. Leon, J. Sawicka, M. Sariahmetoglu, C. J. Schulze, P. G. Scott, D. Szczesna-Cordary, and R. Schulz Degradation of Myosin Light Chain in Isolated Rat Hearts Subjected to Ischemia-Reperfusion Injury: A New Intracellular Target for Matrix Metalloproteinase-2 Circulation, July 26, 2005; 112(4): 544 - 552. [Abstract] [Full Text] [PDF]
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J. A. Simpson, R. Labugger, C. Collier, R. J. Brison, S. Iscoe, and J. E. Van Eyk Fast and Slow Skeletal Troponin I in Serum from Patients with Various Skeletal Muscle Disorders: A Pilot Study Clin. Chem., June 1, 2005; 51(6): 966 - 972. [Abstract] [Full Text] [PDF]
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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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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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R. Labugger, J. A. Simpson, M. Quick, H. A. Brown, C. E. Collier, I. Neverova, and J. E. Van Eyk Strategy for Analysis of Cardiac Troponins in Biological Samples with a Combination of Affinity Chromatography and Mass Spectrometry Clin. Chem., June 1, 2003; 49(6): 873 - 879. [Abstract] [Full Text] [PDF]
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C. J. Schulze, W. Wang, W. L. Suarez-Pinzon, J. Sawicka, G. Sawicki, and R. Schulz Imbalance Between Tissue Inhibitor of Metalloproteinase-4 and Matrix Metalloproteinases During Acute Myoctardial Ischemia-Reperfusion Injury Circulation, May 20, 2003; 107(19): 2487 - 2492. [Abstract] [Full Text] [PDF]
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R. L. Winslow and M. S. Boguski Genome Informatics: Current Status and Future Prospects Circ. Res., May 16, 2003; 92(9): 953 - 961. [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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C. Neagoe, M. Kulke, F. del Monte, J. K. Gwathmey, P. P. de Tombe, R. J. Hajjar, and W. A. Linke Titin Isoform Switch in Ischemic Human Heart Disease Circulation, September 10, 2002; 106(11): 1333 - 1341. [Abstract] [Full Text] [PDF]
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J. T. Colston, B. Chandrasekar, and G. L. Freeman A Novel Peroxide-induced Calcium Transient Regulates Interleukin-6 Expression in Cardiac-derived Fibroblasts J. Biol. Chem., June 21, 2002; 277(26): 23477 - 23483. [Abstract] [Full Text] [PDF]
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P. Eaton, W. Fuller, and M. J. Shattock S-Thiolation of HSP27 Regulates Its Multimeric Aggregate Size Independently of Phosphorylation J. Biol. Chem., June 7, 2002; 277(24): 21189 - 21196. [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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Z. Papp, J. Barta, and G. J.M. Stienen Troponin I Degradation and Myocardial Stunning Circulation, December 18, 2001; 104 (25): e157 - e157. [Full Text] [PDF]
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R. A. Kloner and R. B. Jennings Consequences of Brief Ischemia: Stunning, Preconditioning, and Their Clinical Implications: Part 1 Circulation, December 11, 2001; 104(24): 2981 - 2989. [Abstract] [Full Text] [PDF]
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C. Stamm, I. Friehs, D. B. Cowan, H. Cao-Danh, S. Noria, M. Munakata, F. X. McGowan Jr., and P. J. del Nido Post-ischemic PKC inhibition impairs myocardial calcium handling and increases contractile protein calcium sensitivity Cardiovasc Res, July 1, 2001; 51(1): 108 - 121. [Abstract] [Full Text] [PDF]
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H. Kogler, D. G. Soergel, A. M. Murphy, and E. Marban Maintained contractile reserve in a transgenic mouse model of myocardial stunning Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2623 - H2630. [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. Feng, B. J. Schaus, J. A. Fallavollita, T.-C. Lee, and J. M. Canty Jr Preload Induces Troponin I Degradation Independently of Myocardial Ischemia Circulation, April 24, 2001; 103(16): 2035 - 2037. [Abstract] [Full Text] [PDF]
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G. A. MacGowan, C. Du, D. B. Cowan, C. Stamm, F. X. McGowan, R. J. Solaro, A. P. Koretsky, and P. J. Del Nido Ischemic dysfunction in transgenic mice expressing troponin I lacking protein kinase C phosphorylation sites Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H835 - H843. [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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A. S. Jaffe, J. Ravkilde, R. Roberts, U. Naslund, F. S. Apple, M. Galvani, and H. Katus It's Time for a Change to a Troponin Standard Circulation, September 12, 2000; 102(11): 1216 - 1220. [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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B. E. Sobel and M. M. LeWinter Ingenuous interpretation of elevated blood levels of macromolecular markers of myocardial injury: a recipe for confusion J. Am. Coll. Cardiol., April 1, 2000; 35(5): 1355 - 1358. [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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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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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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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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