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1
Department of Pathology, Vanderbilt University School of Medicine, 4605 TVC, Nashville, TN 37232-5310.
2 Department of Laboratory Medicine and Pathology,
Hennepin County Medical Center, University of Minnesota School of
Medicine, Minneapolis, MN 55415.
a Author for correspondence. Fax 615-343-8420; e-mail bodorgs{at}ctrvax.vanderbilt.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: heart disease • polymyositis • muscular dystrophy • immunohistochemistry • Western blot • ELISA
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After the initial commercial availability of a cTnT immunoassay, investigators believed that cTnT could be detected only during an MI (2). Ongoing studies, however, have established that cTnT can also be released during unstable angina (4)(5)(6), although the amount released, as indicated by the peak serum concentration of cTnT, is usually less than that seen during an MI. This observation led to recognition of the condition "minor myocardial damage" (7). Now, however, a growing number of reports also cite low concentrations of cTnT in sera of patients without any clinically recognizable cardiac disease or injury (8)(9)(10). In one study, 27% of 30 patients with polymyositis (PM) or Duchenne muscular dystrophy (DMD) had above-normal concentrations (>0.25 µg/L) of cTnT in their blood in the absence of evidence of ischemic myocardial disease (8). Elsewhere, 46% of 67 blood samples collected from kidney dialysis patients contained increased (>0.1 µg/L) concentrations of cTnT even when no cardiac muscle injury could be detected (11). The reasons for these findings are unknown, although several explanations have been proposed (12)(13), e.g., reported cross-reactivity of the commercial cTnT immunoassay with skeletal TnT (14), and the presence in these patients of minor myocardial damage that is otherwise unrecognizable by clinical means. The latter, while theoretically possible, is statistically unlikely (i.e., that one- to two-thirds of patients with skeletal muscle disease or chronic renal failure would have minor cardiac damage undetectable by any other currently available diagnostic method).
Another, more likely, possibility is that cTnT is expressed by skeletal muscle during regenerative processes. The physiological basis for this phenomenon is the fact that cTnT is the first TnT isoform seen in developing embryonic and fetal skeletal muscle (15)(16). Reexpression of cTnT in regenerating or diseased skeletal muscle thus would be analogous to reexpression of the B gene of the creatine kinase (CK) enzyme, the early developmental form of CK, during skeletal muscle regeneration (17)(18).
Here we report the results of our investigation into cTnT composition of human skeletal muscle with immunohistochemistry techniques, Western blot analysis, and a commercially available cTnT immunoassay. Using these methods, we have demonstrated the presence of cTnT in biopsy samples of human fetal skeletal muscle and in diseased and nondiseased adult skeletal muscle. The present study parallels, in the following respects, our previously published study describing the composition of cardiac troponin I (cTnI) in normal and diseased skeletal muscle (19): The same diseased patients' samples were analyzed as in the earlier study, and similar techniques (immunohistochemistry and quantitative analysis of the same muscle extracts) were used. The results of the two studies thus are directly comparable because possible between-patient variability in expression of cTnT and cTnI has been eliminated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Diseased skeletal muscle biopsy samples were collected from patients with suspected regenerative skeletal muscle disorders as part of the clinical diagnostic work-up. After the clinical diagnosis was established, residual tissue samples from 13 patients with PM and 6 patients with DMD were stored frozen at -80 °C until immunohistochemistry or extraction of contractile proteins.
All samples were collected in accordance with the Helsinki Declaration of 1975 as revised in 1983, and the study was approved by the appropriate Institutional Review Board guidelines of the participating institutions.
anti-ctnt antibody for immunohistochemistry
Affinity-purified polyclonal goat anti-cTnT antibody (G136-C;
Fortron BioScience, Morrisville, NC) was developed against N-terminal
amino acids 3–15 of human cTnT (20), the sequence
Ile-Glu-Glu-Val-Val-Glu-Glu-Tyr-Glu-Glu-Glu-Glu-Gln. This antibody
reportedly cross-reacts by <0.4% with skeletal TnT isoforms. Although
we attempted to obtain an anti-cTnT-specific monoclonal antibody (M7;
Boehringer Mannheim, Indianapolis, IN) that is reported to have no
measurable cross-reactivity with skeletal TnT isoforms
(14), it was not available for our experimentation.
western blot
Frozen tissue (40–60 mg of normal human myocardium, normal and
diseased skeletal muscle) was homogenized in 1 mL of 200 mmol/L
K2HPO4 buffer (containing 5 mmol/L EGTA, 5
mmol/L ß-mercaptoethanol, and 100 mL/L glycerol) with a Polytron
homogenizer (Brinkmann Instruments, Westbury, NY). The homogenates were
incubated at ambient temperature for 1 h and centrifuged at
20 000g for 30 min, after which aliquots were analyzed for
total protein concentration by a modified Lowry method with
commercially available reagents (cat. no. 690-A; Sigma Diagnostics, St.
Louis, MO). We then electrophoresed 2–10 µg of the homogenates on
sodium dodecyl sulfate–12% polyacrylamide minigels for 1 h at
150 V with MiniProtean Electrophoresis apparatus (Bio-Rad, Hercules,
CA). After electrophoresis, we used a Mini TransBlot electrophoresis
transfer apparatus (Bio-Rad) to transfer the proteins from the gel to a
nitrocellulose membrane, transferring the proteins in the cold for
1 h at 100 V in transfer buffer (25 mmol/L Tris, 192 mmol/L
glycine, and 200 mL/L methanol, pH 8.3). Nonspecific binding sites were
blocked by incubating the membrane overnight at 4 °C with 50 g/L
nonfat dry milk in a buffer of 20 mmol/L Tris base plus 137 mmol/L
NaCl, pH 7.6 (Tris-buffered saline; TBS).
After washing the protein-loaded membrane 3 times in TBS containing 1 mL/L Tween 20, we incubated it for 2 h with the primary anti-cTnT antibody (JS-2 monoclonal anti-cTnT antibody; Lakeland Biomedical, Eden Prairie, MN) diluted to 2 mg/L in 10 g/L dry milk in TBS. We then washed the membrane 3 times in TBS, incubated it for 1 h with a 1:3000 dilution of the secondary horseradish peroxidase-labeled anti-mouse IgG antibody (Amersham, Arlington Heights, IL), and again washed it 3 times in TBS. Finally, we incubated the membrane for 1 min with the chemiluminescent substrate (ECL; Amersham), drained away the substrate solution, placed the membrane in a zip-lock plastic bag, and exposed this to x-ray film (XAR-5; Eastman Kodak, Rochester, NY) for 1 min. Commercially prepared molecular mass markers (Bio-Rad) and purified cTnT and fast skeletal TnT (Spectral Diagnostics, Toronto, Canada) were included in each run. The blot was imaged by using a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA).
in situ ctnt immunohistochemistry
Frozen pieces of patients' and control samples were embedded in
Tissue-Tek OCT (Miles Inc., Diagnostics Div., Elkhart, IN), covered
with talcum powder, and quick-frozen in liquid nitrogen. Sections 7–9
µm thick were cut with a cryostat (Bright Instrument Co., Huntington,
UK), and the slices of tissue were transferred to microscope slides.
The sections were quenched for 20 min with a mixture of methanol/30%
H2O2 (100/1 by vol) and blocked with 50
g/L bovine serum albumin (BSA) solution. Goat anti-cTnT antibody
(G136-C) was used as the first antibody at 15 mg/L in 0.05 mol/L Tris,
9 g/L NaCl, pH 7.2, containing 10 g/L BSA (BSA-TBS); this was pipetted
onto the microscope slides and then incubated for 20–24 h at ambient
temperature in a humidity chamber. For the negative controls, we
substituted nonimmune goat IgG (cat. no. I-5256; Sigma) for the G136-C
antibody. After rinsing off the first antibody with
phosphate-buffered saline, pH 7.2, we added to each slide
150-fold-diluted rabbit anti-goat IgG–peroxidase conjugate (cat. no.
A-4174; Sigma) in BSA-TBS and incubated this for 90 min. We then
removed the second antibody by washing with phosphate-buffered saline
and added 0.91 g/L (final concentration) 3,3'-diaminobenzidine color
reagent (cat. no. D-9015; Sigma). The color was developed for 10–15
min under visual inspection. All slides were counter-stained with
hematoxylin.
assessment of g136-c antibody specificity
Recombinant human cardiac troponin T (rcTnT) for these experiments
was provided by Lillian Lee (Spectral Diagnostics, Toronto, Canada).
For an independent confirmation of G136-C antibody specificity, we
carried out competition assays between soluble and tissue-bound cTnT
antigens for limited antibody-binding capacity as follows. After mixing
stock solutions of rcTnT with 15 mg/L G136-C anti-cTnT antibody in
BSA-TBS to give 8- and 25-fold molar excess of rcTnT, we preincubated
this rcTnT–Ab mixture at 4 °C overnight before pipetting it onto
frozen sections of tissues from normal human heart or diseased skeletal
muscle. We detected binding of G136-C antibody to cTnT localized in
tissue by immunohistochemistry (see above). For control experiments (no
competition), we mixed antibody solutions with rcTnT storage buffer
that contained no rcTnT.
quantification of ctnt and ck-mb in tissue
We homogenized 30 mg of adult human skeletal or cardiac muscle
tissue in 3 mL of 0.05 mol/L Tris buffer (pH 7.4), incubated this for
1 h at 4 °C, and then centrifuged at 100 000g for
1 h; the supernatant liquid was saved and the pellet was
resuspended in the Tris buffer. This process was repeated two more
times to extract the cytosol. The three aliquots containing the cytosol
fraction were pooled and stored at -35 °C until CK-MB and cTnT
could be measured. The pellet remaining after the last centrifugation
was resuspended in 3 mL of 8 mol/L urea buffer (pH 8.0), incubated for
1 h at ambient temperature, and centrifuged at 20 000g
for 30 min. Again, the supernatant liquid was saved and the pellet was
incubated two more times in the urea buffer to extract the myofibrillar
fraction. The three aliquots of myofibrillar fraction in urea buffer
were pooled and stored at -35 °C before performing CK-MB and cTnT
immunoassays.
cTnT concentrations were measured with a commercially available enzyme immunoassay, CardiAC Troponin T, on the ES300 analyzer (both from Boehringer Mannheim). This assay uses streptavidin-coated tubes and two anti-cTnT antibodies. The capture antibody (M7) is specific for cTnT, but the horseradish peroxidase-labeled second antibody (1B10) cross-reacts by ~10% with skeletal muscle TnT, resulting in 2–4% assay cross-reactivity with skeletal TnT (14).
CK-MB mass was quantified with the Baxter Stratus II analyzer and commercially available reagents (all from Dade International, Miami, FL).
Total protein concentration of the extracts was measured by the Lowry method as described above (see Western blot). Concentrations of cTnT and CK-MB are reported as mg/g total protein.
statistics
Statistical differences for the distribution of CK-MB and cTnT
concentrations in the different skeletal muscle groups were determined
by using nonparametric statistics: the Mann–Whitney U-test
and the StatView program for the Macintosh (Abacus Concepts, Berkeley,
CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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G136-C antibody specificity.
G136-C antibody reacted
with cTnT found in human heart tissue as indicated by brown color in
Fig. 1
A. The antibody binding was diminished by increasing amounts of
soluble rcTnT, as depicted by the less-intense color development on
Fig. 1C
and D, corresponding to molar antigen excess of ~8- and
25-fold, respectively, over the G136-C antibody. The latter excess of
rcTnT diminished antibody binding (Fig. 1D
) and produced no brown
color, similar to the negative control (Fig. 1B
).
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In situ cTnT immunohistochemistry.
Sufficient amounts of
muscle tissue were available from five of the six DMD patients for us
to perform immunohistochemistry. Fig. 2
depicts the results of the cTnT immunohistochemistry, each
panel corresponding to one patient. Apparently, not all muscle fibers
are stained by the G136-C antibody, thus indicating expression of cTnT
by some but not all muscle fibers. The ratio of fibers expressing
(brown) and not expressing (lack of color development) cTnT varies
between patients. Furthermore, the intensity of color development can
be seen to vary between individual fibers of the same patient,
suggesting an uneven expression of cTnT.
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All 13 PM patients had sufficient biopsy tissue for
immunohistochemistry. Samples from five PM patients showed no color
development, indicating no cTnT expression (representative results are
shown in Fig. 3
H–J). However, cTnT was detected in tissue samples from the
other PM patients (Fig. 3A
–G, representative samples). As with the DMD
patients, not all muscle fibers exhibited cTnT expression. However,
fewer fibers from PM patients' muscle expressed cTnT and the color
intensity was lighter, suggesting lower expression and concentration of
cTnT in PM muscle than in muscle tissue from DMD patients. All
immunohistochemistry slides lacked brown color development when
nonimmune goat serum was substituted for the G136-C antibody (negative
controls, data not shown).
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Skeletal muscle tissue collected at autopsy from patients who had no
known muscle disease were tested by immunohistochemistry for the
presence or absence of cTnT. The tissue donors had no clinical signs of
chronic renal failure, a condition known to increase cTnT concentration
in blood even in the absence of cardiac muscle damage. No cTnT was
detected in normal vastus lateralis, deltoid, or quadriceps femoris
muscle by immunohistochemistry under the experimental conditions
described in Materials and Methods (results not shown).
However, one normal tissue sample from the rectus abdominis muscle of
one autopsy sample (Fig. 4
D), and the samples from the diaphragm of two other autopsies
(Fig. 4E
and F) developed color after reaction with the G136-C
antibody, indicating the presence of cTnT in these nondiseased skeletal
muscle samples. As with the DMD and PM patients, some but not all
fibers contained cTnT. In diaphragm tissue, up to 20% of the skeletal
muscle fibers developed color, indicating the presence of cTnT.
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To better understand the physiological basis of cTnT distribution in
skeletal muscle, we assayed fetal cardiac and skeletal muscle tissue
from therapeutic abortions (gestational ages 74–135 days). All heart
muscle and skeletal muscle samples exhibited strong staining by the
G136-C antibody, indicating the presence of cTnT in developing human
heart and skeletal muscle (Fig. 4
, A–C).
Western blot.
Fig. 5
shows representative Western blot analyses of normal human
cardiac muscle, normal human skeletal muscle, and diseased human
skeletal muscle for cTnT. Expression of comigrating multiple isoforms
of cTnT was demonstrated in both heart (lane 2) and diseased skeletal
muscle obtained from DMD patients (lane 4); both comigrated with the
cTnT standard (lane 1) and showed molecular masses ranging from 33 to
39 kDa. Western blot detected no cTnT in normal skeletal muscle control
(lane 3), purified human skeletal muscle TnT (lane 5), purified human
CK-MB (lane 6), or purified human cTnI (data not shown).
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Quantification of cTnT and CK-MB in adult tissue.
Results of quantitative analyses of muscle tissue extracts are
presented in Table 1
. The mean CK-MB concentration in extracts of four normal
skeletal muscle samples was 1.3 mg/g total protein. In the extracts of
skeletal muscle from 13 PM and 6 DMD patients, mean CK-MB
concentrations were 0.76 and 9.0 mg/g total protein, respectively. Mean
cTnT concentrations in extracts of skeletal muscle from normal, PM, and
DMD patients were 0.8, 0.73, and 4.37 mg/g total protein, respectively.
Nonparametric statistical analysis indicated that CK-MB and cTnT
content of human heart muscle and diseased (DMD) skeletal muscle
extracts was significantly greater (P <0.001) than in
normal (nondiseased) skeletal muscle (Table 1
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Utilizing the confirmed specific G136-C antibody, we demonstrated the
expression of cTnT in developing, diseased (PM and DMD), and,
occasionally, normal (nondiseased) human skeletal muscle. The single
fiber that stained with the G136-C anti-cTnT antibody in normal
(control) skeletal muscle is significantly smaller than the other
fibers (Fig. 4D
). The fact that its appearance differs from that of the
surrounding normal fibers suggest a possible degenerating fiber in
otherwise healthy muscle. This result demonstrates that cTnT can be
present in otherwise "normal" skeletal muscle tissue. As for the
patients' samples, cTnT was expressed by the muscle tissue of all five
DMD patients we examined, but only 8 of the 13 PM patients' skeletal
muscle contained cTnT by immunohistochemistry. The staining pattern
implied a nonuniform expression of cTnT by the involved muscle. Only
certain muscle fibers demonstrated the presence of cTnT, but this
staining was consistent: On serial sections of the muscle tissue,
always the same fibers exhibited color development (data not shown).
The observed staining pattern, i.e., staining of individual muscle
fibers but not the whole section, is also consistent with a specific
reaction between the G136-C antibody and its antigen, cTnT.
Nonspecific reaction was further ruled out by assaying the negative controls, which contained nonimmune goat IgG instead of the anti-cTnT G136-C antibody. Cross-reaction of the G136-C antibody with skeletal TnT as the reason for the observed staining pattern is unlikely. If G136-C antibody did react with skeletal TnT, one should observe the staining of all skeletal muscle fibers. Staining of some but not all fibers could happen only if a fraction—not all—of the muscle fibers contained skeletal TnT, which is physiologically impossible.
Independent of immunohistochemical staining, Western blot analysis with a monoclonal anti-cTnT antibody (JS-2) clearly demonstrated multiple cTnT isoform expression (molecular masses 33–39 kDa) in DMD patients, consistent with reexpression of cTnT isoforms identified in failing hearts (24). PM skeletal muscles were not studied by Western blot.
Using the ES300 CardiAC Troponin T commercial immunoassay, we detected measurable amounts of cTnT in the tissue extracts from the adult skeletal muscle biopsy samples, including the normal (nondiseased) human skeletal muscles. Unfortunately, this FDA-approved cTnT immunoassay has measurable cross-reactivity with skeletal TnT (14), which makes it impossible to tell whether the measured cTnT in the skeletal muscle extracts is pure cTnT or is the sum of assay cross-reactivity with skeletal TnT and cTnT. As reported elsewhere (14), the ES300 cTnT immunoassay cross-reacts 2% with skeletal TnT; however, this reported cross-reactivity cannot account for the observed cTnT concentration in the muscle extracts (0.8 and 4.37 mg/g protein, on average, in nondiseased and DMD muscle, respectively). For the apparent cTnT concentration to be due only to skeletal TnT at 2% assay cross-reactivity, ~4–22% of all muscle proteins should be skeletal TnT. Therefore, although some cross-reactivity may have contributed to the signal in the cTnT immunoassay, a substantial amount of cTnT would have to be present in skeletal muscle in addition to skeletal TnT. Incidentally, because the ES300 cTnT immunoassay utilizes different antibodies than those used for immunohistochemistry and Western blot analyses, the ES300 results should be considered independent confirmation of the presence of cTnT in adult diseased and nondiseased skeletal muscle.
[After this study was completed, Boehringer Mannheim reported the development of an improved cTnT immunoassay that had no measurable cross-reactivity with skeletal TnT; however, this assay was not available for our experimentation, and what the cTnT concentrations in our samples would be with this improved assay remains to be seen. Interestingly, a recent report (25) suggested the presence of cTnT in sera of patients with renal failure even when assayed with the improved cTnT immunoassay.]
An increasing body of literature has documented the presence of cTnT in blood of patients who have had no clinical evidence of myocardial damage (8)(9)(10)(11)(13). Unexplained increases in cTnT, i.e., increases without clinically detectable myocardial injury, were reported in 27% to 78% of all patients examined by various authors. These cTnT increases were frequently but not always accompanied by increases in CK-MB. To further complicate this picture, the various authors used markedly different cTnT concentrations (ranging from 0.1 to 0.5 µg/L, depending on the immunoassay system used) to identify false-positive cTnT increases. Retrospective evaluation of these patients sometimes revealed previously undetected myocardial injury, but a substantial proportion of patients with increased cTnT blood concentration revealed no cardiac origin. Possible assay cross-reactivity with skeletal TnT, or cTnT expression by skeletal muscle, or release of cTnT by skeletal muscle after an injury have been proposed as plausible explanations (13). Here, we have demonstrated the presence of cTnT in regenerating and normal human skeletal muscle; i.e., cTnT is present and therefore can be released from skeletal muscle (diseased or nondiseased). Thus, low concentrations of cTnT in blood are not necessarily indications of cardiac muscle injury.
Using immunohistochemistry, we also demonstrated the presence of cTnT in human fetal skeletal muscle tissues. These findings are consistent with the results of previous animal studies and confirm that cTnT is the predominant early developmental TnT isoform, even in human skeletal muscle (15)(26). cTnT transcription and protein expression decreases in skeletal muscle after birth, but cTnT continues to be the major TnT isoform in adult heart (27). In this respect, the physiology of cTnT resembles that of the B subunit of CK, both being the early developmental form expressed in all muscles (17). Increased concentrations of CK-MB, as well as of myosin light chain 1 (MLC-1), have been demonstrated by immunohistochemistry in regenerating human skeletal muscle (28), and in adults both of these proteins are ordinarily produced predominantly in cardiac muscle. However, both proteins were also found in striated muscle fibers within malignant teratomas (28) and in diseased (regenerating) adult skeletal muscle, thus indicating reexpression of early genes during regenerative processes. Other investigators, using animal models, have reported similar findings regarding the B gene of CK (18)(29). Like the B subunit of CK, cTnT is reexpressed in human skeletal muscle during regeneration as evidenced by our immunohistochemistry, Western blot, and quantitative biochemical analyses.
An unexpected finding of our study was the occasional detection of cTnT
in normal skeletal muscle. As demonstrated in Fig. 4
, cTnT expression
by normal muscle tissue varies, depending on the muscle group. The
greatest numbers of fibers expressing cTnT were found in diaphragm.
Again, this phenomenon is similar to CK-MB expression by skeletal
muscle, in that, aside from myocardium, the largest CK-MB concentration
is found in diaphragm (30). Our study shows that the
general pattern of the diaphragm's retaining expression or
reexpression of early developmental proteins by skeletal muscle during
muscle regeneration is real for cTnT, thereby providing some insight
into the physiology of regulatory protein expression in healthy and
diseased muscle. At present, it is unclear how this knowledge could be
applied to the diagnosis or treatment of various muscle diseases.
cTnT and cTnI are distinct proteins with different physiologies and clinical characteristics, although both are useful markers of myocardial injury. We recently reported the results of similar immunohistochemistry studies investigating cTnI expression by normal and diseased skeletal muscle (19). The muscle biopsy samples from the PM and DMD patients analyzed by cTnI immunohistochemistry and reported there are the same as the samples reported here, and none contained detectable cTnI (19). In contrast, all five DMD samples and 8 of 13 PM samples contained detectable amounts of cTnT. Because the conditions for both stainings are very similar, we do not believe that the negative staining for cTnI vs the positive staining for cTnT in the same muscle biopsy samples are technical artifacts. We used similar primary and secondary antibody concentrations, similar incubation times and temperature, identical concentrations of the color reagent substrate, and similar times for color development throughout the two experiments.
Therefore, we believe our results help explain the findings of other investigators who detected no cTnI in the blood of patients with clinical conditions commonly associated with skeletal muscle regeneration unless those patients also had cardiac muscle injury (31)(32). Few studies have been published comparing cTnI and cTnT blood concentrations in the same group of patients. Hafner et al. found that 46% of 67 specimens from patients with chronic renal failure had increased cTnT, but only 1 patient from the same group had increased cTnI (11). Upon further investigation, the only patient with increased cTnI had a documented history of cardiovascular disease, but none of the patients with increases in only cTnT presented evidence of cardiac injury, even when examined by echocardiography. In another study by Li et al. (33), 7of 79 patients (8.9%) with end-stage renal disease had detectable cTnI (>0.35 µg/L), but only 4 of these had a cTnI concentration indicative of myocardial infarction (>1.5 µg/L). Subsequently, 2 of these 4 patients were recognized to have had a clinically confirmed non-Q-wave infarction. In contrast, >63% of the 79 patients had above-normal cTnT concentrations, according to the kit's manufacturer's cutoff value (0.1 µg/L cTnT). Even if a higher cutoff (0.2 µg/L cTnT) were applied, as recommended by several authors, >46% of all renal failure patients would have had above-normal cTnT. Patients with end-stage renal disease are known to have myopathy with muscle regeneration (34), and here we have demonstrated cTnT expression by regenerating skeletal muscle. Our immunohistochemistry experiments may provide an explanation for why so many patients with renal failure have increased cTnT but not cTnI in their blood.
Our findings demonstrate that the absolute cardiac specificity of cTnT must be revised. Thus, the clinical significance of an increased serum cTnT concentration requires cautious interpretation. We demonstrate that cTnT can be present and therefore can be released from skeletal muscle and thereby lead to increased serum cTnT concentrations without cardiac muscle injury. What false impressions this fact may present in clinical practice needs to be evaluated with appropriately designed clinical outcome studies.
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  P. O Collinson and D. C Gaze Cardiac troponins in patients with renal failure: what are we measuring and when should we measure it? Ann Clin Biochem, July 1, 2009; 46(4): 269 - 270. [Full Text] [PDF]
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 J S Hamilton and P C Sharpe Two cases of inflammatory muscle disease presenting with raised serum concentrations of troponin T J. Clin. Pathol., December 1, 2005; 58(12): 1323 - 1324. [Abstract] [Full Text] [PDF]
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 L. Babuin and A. S. Jaffe Troponin: the biomarker of choice for the detection of cardiac injury Can. Med. Assoc. J., November 8, 2005; 173(10): 1191 - 1202. [Abstract] [Full Text] [PDF]
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 R. Rej Clinical Chemistry through Clinical Chemistry: A Journal Timeline Clin. Chem., December 1, 2004; 50(12): 2415 - 2458. [Abstract] [Full Text] [PDF]
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 Y Sato, T Kita, Y Takatsu, and T Kimura Biochemical markers of myocyte injury in heart failure Heart, October 1, 2004; 90(10): 1110 - 1113. [Abstract] [Full Text] [PDF]
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 S Clark, P Newland, C W Yoxall, and N V Subhedar Concentrations of cardiac troponin T in neonates with and without respiratory distress Arch. Dis. Child. Fetal Neonatal Ed., July 1, 2004; 89(4): F348 - F352. [Abstract] [Full Text] [PDF]
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 K. B. Wallace, E. Hausner, E. Herman, G. D. Holt, J. T. Macgregor, A. L. Metz, E. Murphy, I.Y. Rosenblum, F. D. Sistare, and M. J. York Serum Troponins as Biomarkers of Drug-Induced Cardiac Toxicity Toxicol Pathol, January 1, 2004; 32(1): 106 - 121. [PDF]
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 M. Panteghini Acute Coronary Syndrome: Biochemical Strategies in the Troponin Era Chest, October 1, 2002; 122(4): 1428 - 1435. [Abstract] [Full Text] [PDF]
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S. Fredericks, J. F. Murray, N. D. Carter, A. M.S. Chesser, S. Papachristou, M. M. Yaqoob, P. O. Collinson, D. Gaze, and D. W. Holt Cardiac Troponin T and Creatine Kinase MB Content in Skeletal Muscle of the Uremic Rat Clin. Chem., June 1, 2002; 48(6): 859 - 868. [Abstract] [Full Text] [PDF]
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S. Fredericks, J. F. Murray, M. Bewick, R. Chang, P. O. Collinson, N. D. Carter, and D. W. Holt Cardiac Troponin T and Creatine Kinase MB Are Not Increased in Exterior Oblique Muscle of Patients with Renal Failure Clin. Chem., June 1, 2001; 47(6): 1023 - 1030. [Abstract] [Full Text] [PDF]
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D. S. Ooi, D. Zimmerman, J. Graham, and G. A. Wells Cardiac Troponin T Predicts Long-Term Outcomes in Hemodialysis Patients Clin. Chem., March 1, 2001; 47(3): 412 - 417. [Abstract] [Full Text] [PDF]
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A. Hammerer-Lercher, P. Erlacher, R. Bittner, R. Korinthenberg, D. Skladal, S. Sorichter, W. Sperl, B. Puschendorf, and J. Mair Clinical and Experimental Results on Cardiac Troponin Expression in Duchenne Muscular Dystrophy Clin. Chem., March 1, 2001; 47(3): 451 - 458. [Abstract] [Full Text] [PDF]
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 Y. Sato, T. Yamada, R. Taniguchi, K. Nagai, T. Makiyama, H. Okada, K. Kataoka, H. Ito, A. Matsumori, S. Sasayama, et al. Persistently Increased Serum Concentrations of Cardiac Troponin T in Patients With Idiopathic Dilated Cardiomyopathy Are Predictive of Adverse Outcomes Circulation, January 23, 2001; 103(3): 369 - 374. [Abstract] [Full Text] [PDF]
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D. Wayand, H. Baum, G. Schatzle, J. Scharf, and D. Neumeier Cardiac Troponin T and I in End-Stage Renal Failure Clin. Chem., September 1, 2000; 46(9): 1345 - 1350. [Abstract] [Full Text] [PDF]
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S. Fredericks, P. O. Collinson, D. W. Holt, P. A. Isotalo, D. C. Greenway, and J. G. Donnelly Response to ""Increased Creatine Kinase MB and Cardiac Troponin T with Normal Cardiac Troponin I in Metastatic Alveolar Rhabdomyosarcoma"" • The authors of the Letter cited above reply: Clin. Chem., March 1, 2000; 46(3): 432 - 435. [Full Text] [PDF]
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V. Ricchiuti and F. S. Apple RNA Expression of Cardiac Troponin T Isoforms in Diseased Human Skeletal Muscle Clin. Chem., December 1, 1999; 45(12): 2129 - 2135. [Abstract] [Full Text] [PDF]
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A. H.B. Wu, F. S. Apple, W. B. Gibler, R. L. Jesse, M. M. Warshaw, and R. Valdes Jr. National Academy of Clinical Biochemistry Standards of Laboratory Practice: Recommendations for the Use of Cardiac Markers in Coronary Artery Diseases Clin. Chem., July 1, 1999; 45(7): 1104 - 1121. [Abstract] [Full Text] [PDF]
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V. Ricchiuti, E. M. Voss, A. Ney, M. Odland, P. A. W. Anderson, and F. S. Apple Cardiac troponin T isoforms expressed in renal diseased skeletal muscle will not cause false-positive results by the second generation cardiac troponin T assay by Boehringer Mannheim Clin. Chem., September 1, 1998; 44(9): 1919 - 1924. [Abstract] [Full Text] [PDF]
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D. S. Ooi and A. A. House Cardiac troponin T in hemodialyzed patients Clin. Chem., July 1, 1998; 44(7): 1410 - 1416. [Abstract] [Full Text] [PDF]
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C. Haller, J. Zehelein, A. Remppis, M. Muller-Bardorff, and H. A. Katus Cardiac troponin T in patients with end-stage renal disease: absence of expression in truncal skeletal muscle Clin. Chem., May 1, 1998; 44(5): 930 - 938. [Abstract] [Full Text] [PDF]
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C. Haller, H. A. Katus, F. S. Apple, and S. W. Sharkey Expression of Cardiac Troponin T in Skeletal Muscle Clin. Chem., February 1, 1998; 44(2): 358 - 359. [Full Text] [PDF]
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S. Fredericks, G. Merton, M.-J. Lerena, D. W. Holt, G. S. Bodor, F. S. Apple, and E. M. Voss Markers of Myocardial Damage Clin. Chem., February 1, 1998; 44(2): 362 - 365. [Full Text] [PDF]
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H. Baum, S. Braun, W. Gerhardt, G. Gilson, G. Hafner, M. Muller-Bardorff, W. Stein, G. Klein, C. Ebert, K. Hallermayer, et al. Multicenter evaluation of a second-generation assay for cardiac troponin T Clin. Chem., October 1, 1997; 43(10): 1877 - 1884. [Abstract] [Full Text] [PDF]
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M. D. McLaurin, F. S. Apple, E. M. Voss, C. A. Herzog, and S. W. Sharkey Cardiac troponin I, cardiac troponin T, and creatine kinase MB in dialysis patients without ischemic heart disease: evidence of cardiac troponin T expression in skeletal muscle Clin. Chem., June 1, 1997; 43(6): 976 - 982. [Abstract] [Full Text] [PDF]
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P. O. Collinson To T or Not to T, That Is the Question Clin. Chem., March 1, 1997; 43(3): 421 - 423. [Full Text] [PDF]
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