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Articles |
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Analytical Unit, Cardiological Sciences, and
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Medical Genetics Unit, St. George’s Hospital Medical School, London SW17 0RE, United Kingdom; and Departments of
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Renal Medicine and
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Chemical Pathology, St. George’s Hospital, London SW17 0QT, United Kingdom.
aAuthor for correspondence. Fax 44-20-8767-9687; e-mail frederic{at}sghms.ac.uk.
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Abstract |
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Methods: Skeletal muscle (exterior oblique) biopsies were taken from healthy living kidney donors (n = 5) and transplant recipients (n = 19). The amounts of cTnT and creatine kinase (CK) isoenzymes in skeletal muscle of healthy controls were compared with those in patients with renal failure (Wilcoxon–Mann–Whitney test). cTnT was measured quantitatively by a second-generation assay, with a limit of detection of 1 µg/g of protein, and qualitatively by immunohistochemistry and immunoblotting. CK-MB was measured by quantitative electrophoresis.
Results: Minute quantities of cTnT were detected in 2 of the 5 (40%) control samples and 9 of the 19 (47%) renal failure samples, respectively, at mean concentrations of <5 µg/g of protein for both subject groups. This was <1/6000th that found in heart muscle. There was no significant difference in cTnT or CK-MB content in skeletal muscle between healthy controls and patients with renal failure. Increased serum cTnT did not predict detectable cTnT in skeletal muscle. cTnT was not detected qualitatively by immunoblotting or immunohistochemistry in any skeletal muscle samples.
Conclusions: Uremia does not affect the content of cTnT or CK-MB in exterior oblique muscle, suggesting that cTnT detected in serum from patients with renal failure does not originate from skeletal muscle.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been suggested that cTnT and CK-MB are expressed by diseased skeletal muscle found in patients with chronic renal failure (CRF) (7). It has been shown that myopathy (8)(9) and impaired exercise tolerances (10)(11) are associated with CRF and dialysis. An electron micrographic study has demonstrated evidence of muscle regeneration in biopsies of skeletal muscle taken from patients with CRF (12).
There is evidence that in regenerating muscle, CK-MB expression is increased (13) and cTnT is expressed; examples include rat skeletal muscle in response to injury and denervation (14), human Duchenne muscular dystrophy (DMD) (15), and the C2C12 mouse myocyte cell line (16).
In cardiac myocytes, cTnT is encoded by one gene, but four possible isoforms may be produced by alternative splicing (17). These isoforms are present in developing skeletal muscle and are down-regulated after birth (18). The B subunit of CK is similarly expressed in relatively larger amounts in developing skeletal muscle. Isoforms of cTnT and increased amounts of CK-MB have also been detected in diseased skeletal muscle (6)(15). If released into the circulation, these isoforms could potentially be detected in serum by the cTnT immunoassays, producing falsely increased or spurious cTnT values, which may be wrongly interpreted as evidence of myocardial damage. This misdiagnosis could be particularly important in patients with CRF with regenerating skeletal muscle. In healthy adult skeletal muscle, these cTnT isoforms are completely down-regulated; thus, the problem of false positives should not arise with adults without skeletal myopathy. The controversy of false positives and cTnT associated with regenerating skeletal muscle has not affected use of cTnI as a marker of myocardial damage. In contrast to both CK-MB and cTnT, Ricchiuti et al. (7) have stated that cTnI is not expressed during any stage of skeletal muscle development. In addition, cTnI immunoreactivity has been shown to be absent from diseased regenerating skeletal muscle (19). For this reason, the subject of cTnI in skeletal muscle has not been addressed in this study.
In patients with CRF, however, it is unclear whether cTnT isoforms detected in serum are derived from skeletal muscle or result from myocardial damage (7). It was, therefore, the aim of this study to relate cTnT detected in skeletal muscle to cTnT detected in circulation. We measured muscle cTnT quantitatively and qualitatively, using two different qualitative techniques. In addition, we investigated the effect of uremia on CK-MB activity in skeletal muscle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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human muscle samples
Samples of the exterior oblique muscle were taken from patients
with ESRD during elective renal transplant surgery. Samples were
obtained over a period of 1 year from patients (n = 19) receiving
either cadaver or live-donor kidneys. Control samples of the same
muscle were obtained from live related kidney donors (n = 5).
Human cardiac tissue was obtained from left atrial appendage (LAA)
samples (n = 6) obtained from explanted hearts taken during
cardiac transplant surgery. The cardiac transplant patients were all
diagnosed as having dilated cardiomyopathy. Muscle samples were split
into two portions, frozen in liquid nitrogen, and stored at -80 °C
until use. Serum samples were obtained from all patients at the time of
renal transplant surgery. Approval was obtained from the local research
ethics committee of St. George’s Hospital, and consent was obtained
from all patients.
The median age of the patients with ESRD (7 males and 12 females) was 51 years (range, 36–65 years). All 19 patients were hypertensive, 6 were diabetic, 8 had hyperlipidemia, 5 smoked, and 2 were ex-smokers.
preparation of muscle samples
Muscle samples were coarsely ground in liquid nitrogen with a
mortar and pestle over dry ice. The coarse powdered muscle was then
split into two aliquots; one aliquot was homogenized for
immunoblotting, and the other was homogenized for the measurement of
tissue content of cTnT, CK, and CK isoenzymes. The other frozen
portion, collected at the time of surgery, was retained for
immunohistochemical analysis.
preparation of cytosolic and myofibrillar fractions for assay of CK
and cTnT
Cytosolic and myofibrillar extracts of muscle samples were
prepared according to the method of Bodor et al. (19).
Approximately 30 mg of coarsely ground muscle powder was homogenized in
3 mL of 0.05 mol/L Tris buffer (pH 7.4) in an Ultra-Turrax homogenizer
(Merck). The homogenate was incubated for 1 h at 4 °C and
centrifuged at 100 000g for 1 h. The supernatant was
retained, and the pellet was resuspended in 3 mL of 0.05 mol/L Tris
buffer. This process was repeated twice to produce a cytosolic
fraction. The three aliquots containing the cytosolic fraction were
then pooled, aliquoted, and stored at -80 °C until analysis for CK,
CK isoenzymes, cTnT, and total protein. The pellet remaining after the
last centrifugation (containing the myofibrillar proteins) was
resuspended in 3 mL of 0.05 mol/L Tris (pH 8.0) containing 8 mol/L
urea, 1 mmol/L CaCl2, and 15 mmol/L
ß-mercaptoethanol. This suspension was incubated for 1 h at room
temperature and then centrifuged at 20 000g for 30 min. The
supernatant was retained, and the pellet was resuspended in 3 mL of the
same buffer. This process was repeated twice more to extract the
myofibrillar fraction. The three aliquots of myofibrillar fraction were
pooled.
Urea was removed from the myofibrillar fraction by use of a PD-10 sample preparation gel exclusion cartridge (Amersham Pharmacia Biotech), in accordance with the manufacturer’s instructions; the eluent was Tris buffer without urea. This solution was then used for the analysis of cTnT, CK, CK isoenzymes, and total protein concentrations.
measurement of CK isoenzymes and cTnT
Total CK activity was measured quantitatively by the
N-acetylcysteine activated (Roche) method on a Cobas Mira
(Roche) analyzer at 37 °C. Cytosolic extracts were used to measure
CK isoenzymes, which were separated and quantified by agarose gel
electrophoresis in the Rapid Electrophoresis system (Helena
Laboratories). cTnT was measured by the second-generation Enzymune
method, performed on an ES300 analyzer (Roche). The lower limit of
detection for this assay has been reported as 0.01 µg/L, with a
functional sensitivity (between-assay CV = 20%) of 0.05–0.1
µg/L (20)(21). This lower limit of detection
is comparable to that of the newer assay in current use (third
generation), although the reproducibility of the newer assay has been
improved at lower concentrations. According to the manufacturer’s
package insert, good correlation has been shown between the second- and
third-generation assays at concentrations of 0.02–20 µg/L
(r = 0.98).
We diluted all heart cytosolic and urea-free myofibrillar fractions
before analysis to a concentration within the range of the calibrators
for each assay, using serum diluent supplied by the assay manufacturer.
The use of this diluent was to reduce matrix effects. Skeletal
cytosolic extracts were diluted 1:3, and skeletal myofibrillar
fractions were diluted 1:2. All skeletal muscle samples were diluted to
protein concentrations 
20 mg/L before cTnT assay. The calculated
limit of detection of the measurement was, thus, 1 µg/g of protein.
Total protein was determined by the Bradford method (22). cTnT was measured in serum samples obtained from all ESRD patients at the time of transplant.
The amounts of cTnT in the skeletal muscle samples from the ESRD and the healthy control groups were compared using a Wilcoxon–Mann–Whitney test. This test was also used to compare the CK-MB content. A Kruskal–Wallis ANOVA was used to compare the cTnT content and the CK-MB content of the three muscle sample types, normal skeletal muscle, ESRD skeletal muscle, and LAA muscle.
immunohistochemistry
Serial 10-µm-thick sections, cut on a cryostat, were mounted on
gelatin-coated slides, air dried, and then stored at -80 °C until
required for immunohistochemistry. Three slides were prepared for each
specimen (with a minimum of five sections per slide). Sections were cut
from biopsy material taken from the exterior oblique muscle of healthy
patients and patients with ESRD; cardiomyopathic LAA muscle samples
were used as positive controls.
Sections were air-dried and then fixed in 40 g/L paraformaldehyde in phosphate-buffered saline (100 mmol/L phosphate, pH 7.4, 8.5 g/L NaCl) for 10 min on ice. The sections were washed in Tris-buffered saline (TBS; 50 mmol/L Tris, pH 7.6, 8.5 g/L NaCl), and endogenous peroxidase was inhibited by incubation for 5 min in 30 mL/L hydrogen peroxide in methanol. After the sections were washed in TBS, they were preincubated for 2 h with 40 mL/L normal horse serum in TBS. For each specimen, the sections of one slide were each incubated overnight at 4 °C with anti-cTnT M7 (1:200), anti-cTnT M11-7 (1:200), or diluent alone (40 mL/L normal horse serum in TBS). The sections were then processed at room temperature using the avidin-biotin complex method. Briefly, after washing between each step, the sections were first incubated with biotinylated horse anti-mouse IgG (1:200 in TBS for 2 h; Vector Laboratories) followed by incubation with the avidin-biotin-peroxidase complex (ABC Elite; Vector Laboratories). Bound peroxidase was then visualized by incubation in 0.08 g/L diaminobenzidine tetrahydrochloride, 1 g/L nickel sulfate, and 0.07 mL/L hydrogen peroxide in 50 mmol/L Tris (pH 7.2) for 10 min. The sections were then dehydrated, cleared in xylene, and mounted in preparation for examination by light microscopy.
gel electrophoresis and immunoblotting of cTnT
Muscle samples were homogenized in phosphate buffer (200 mmol/L,
pH 7.4) containing 100 mL/L glycerol, 5 mmol/L EGTA, and 5 mmol/L
ß-mercaptoethanol in an Ultra-Turrax homogenizer. Tissue homogenates
were then subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Skeletal muscle sample homogenates (15 µg
of total protein) and LAA muscle sample homogenates (2.5 µg of total
protein) were loaded on 10% polyacrylamide minigels and
electrophoresed according to the Laemmli method (23) using
the Xcell II precast gel system (Novex). Size-separated proteins were
then electrophoretically blotted onto nitrocellulose membranes
(Amersham Pharmacia Biotech). After electrophoresis, the membranes were
blocked with 50 g/L powdered nonfat milk in phosphate-buffered saline
overnight at 4 °C. The membranes were then incubated with either of
the anti-cTnT monoclonal antibodies M11-7 or M7 (Roche). The
concentration of all primary antibodies was 2 mg/L. Horseradish
peroxidase conjugated to goat anti-mouse IgG (Dako) was then used at a
dilution of 1:1000 for the detection of specific binding of the
antibodies. All antibodies were diluted in phosphate-buffered saline
containing 50 g/L powdered nonfat milk. Immunoblots were developed with
ECLTM substrate (Amersham Pharmacia Biotech).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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No cTnT was detected by the quantitative assay in 10 of the 19 ESRD
skeletal muscle samples. The mean cTnT concentration in the
myofibrillar fractions was <5 µg/g of protein for both the ESRD and
the control group. cTnT was not detected in any of the cytosolic
fractions. In the myofibrillar fractions, there was no statistically
significant difference in cTnT concentrations between the healthy
controls and the patients with renal failure (P =
0.525). The median (range) cTnT concentrations in the myofibrillar
skeletal muscle samples were 0 (0–9) µg/g of protein for controls
and 0 (0–6) µg/g of protein for ESRD patients. The median (range)
cTnT concentrations in the LAA samples were 38.6 (23.2–81.0) mg/g of
protein in the myofibrillar fractions and 2.5 (2.0–3.6) mg/g of
protein in the cytosolic fractions. A positive signal (
1 µg/g of
protein) for cTnT was measured in 9 of the 19 (47%) skeletal muscle
samples from patients with ESRD. However, these were all <7 µg/g of
protein. The amounts found in the myofibrillar fractions of the LAA
samples were >6000-fold higher than those found in the comparable
skeletal muscle samples. The highest amount of cTnT detected was 6
µg/g of protein; this patient did not have a positive serum cTnT.
Four of the patients had positive serum cTnT results; of these four,
two gave a positive signal of cTnT in skeletal muscle of 1 µg/g of
protein. Two of the five (40%) control skeletal muscle samples also
gave a positive signal for cTnT. The largest signal for cTnT in
skeletal muscle was measured in a muscle sample from a healthy control
(9 µg/g of protein).
The distribution of CK isoenzymes is summarized in Fig. 1
. There were no significant differences in CK-MB content between
the samples from patients with ESRD and the healthy donors
(P = 0.270).
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There was specific sarcomeric staining for cTnT in human cardiac
control tissue, but no positive signal was observed in skeletal muscle
taken from healthy kidney donors or from patients with ESRD with either
of the two antibodies, M7 and M11-7 (Fig. 2
).
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Representative immunoblots of heart, control skeletal muscle, and
skeletal muscle from renal failure patients for the two antibodies
M11-7 and M7 are shown in Fig. 3
. A major band was observed in extracts from LAA samples, which
migrated at a molecular mass of 39 kDa, as detected by both M7 and
M11-7. Positive staining was found in all 6 LAA samples analyzed,
whereas none of the 24 skeletal muscle samples had 40-kDa
(7) bands that were immunoreactive with either of the two
antibodies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In heart, the CK-MB isoenzyme accounts for 15–40% of total CK activity, CK-MM being the predominant isoenzyme (24). However, our data showed that CK-MB is the predominant CK isoenzyme. The reasons for this may be that we analyzed muscle taken only from the LAA and not from ventricular myocardium. The only material that was available to us was the LAA. In left ventricular muscle from an explanted cardiomyopathic heart, we found that the CK-MB component was 27% of total CK (25). Applying the same technique and using animal hearts, we also found large differences in CK isoenzyme distribution between the atria and ventricles of the same heart (data not shown). LAA may not be typical myocardial tissue with regard to CK isoenzyme distribution. However, for the purpose of our experiment, we used the LAA samples as a positive control. The LAA data demonstrated that CK-MB was easily detectable when we used the Rapid Electrophoresis system to analyze striated muscle extracts.
Trace amounts of cTnT were detected in 9 of the 19 skeletal muscle
samples. There was no correlation between the frequency of positive
cTnT in serum of these patients and the presence of detectable amounts
cTnT in skeletal muscle. These detected amounts of cTnT did not give
rise to positive serum results for cTnT. The amounts of cTnT detected
in all skeletal muscle samples by this system are subject to a degree
of inaccuracy at these very low concentrations. The cTnT assay
performed on the ES300 is designed, essentially, for analyzing serum or
plasma samples. There is a substantial matrix effect observed with this
system. In studies to validate this method, we found that Tris buffer
and deionized water gave a positive signal on this system
(25). For this reason, all muscle extracts were diluted in
assay diluent provided by the manufacturer before analysis. In the case
of heart myofibrillar extracts, the samples were diluted by as much as
1:100. Such large dilutions virtually eliminate matrix effects.
Skeletal muscle samples contained 20 mg/L protein from the
myocyte extract. When we applied the reported limit of detection for
this assay of 0.01 µg/L (20), our detection limit for cTnT
was 0.001 mg/g of protein. Serial dilutions of LAA homogenates showed
that the detection limit of the assay was similar to that reported by
Muller-Bardorff et al. (20). All of the positive values
detected were 0.01–0.5 µg/L for the assay of diluted samples. The
finding that the number of "positive" samples was not statistically
different between the ESRD and donor muscle samples suggests that these
positive results may represent analytical inaccuracy or impression. The
highest value (0.4 µg/L) occurred in a control sample. Although some
of the skeletal muscle samples gave a positive signal, this did not
correspond to an increase in circulating cTnT concentrations.
Our conclusion that cTnT is not abnormally expressed in skeletal muscle of patients with ESRD is in agreement with the findings of Haller et al. (26), who found no expression of cTnT at the mRNA or protein level in skeletal muscle in patients with ESRD who had positive serum concentrations of cTnT. Our findings are also in agreement with the conclusions of Ricchiuti and co-workers (7)(27) that the second- and third-generation cTnT assays would detect only cTnT in circulation that originated from cardiac muscle. We did not reproduce the findings of Ricchiuti and co-workers (7)(27) regarding the positive immunoblotting of skeletal muscle of patients with ESRD, using M7 or M11-7. Ricchiuti and co-workers (7)(27) reported a particularly high frequency of positive staining when they used the antibody M11-7. However, the results of our immunohistochemistry and analysis of tissue content by the cTnT assay support our negative immunoblots. Our negative findings using the cTnT assay are also in agreement with the conclusions of Ricchiuti et al. (7) and the observations of Muller-Bardorff et al. (20) that a positive signal produced in the assay format using a combination of the two antibodies, M7 and M11-7, will occur only in response to binding of the adult cTnT isoform.
This study could have been strengthened by the measurement of cTnT expression at the mRNA level. Using reverse transcription-PCR, Messner et al. (28) detected mRNA for cTnI and cTnT in 19% of skeletal muscle biopsies taken from patients with various skeletal myopathies, but found neither cTnI nor cTnT in serum from these patients. Ricchiuti and Apple (27) detected cTnT mRNA in skeletal muscle biopsies from DMD patients at amounts comparable to those found in heart muscle, but they did not detect cTnT protein when they used laser densitometry to quantify Western blots probed with M11-7 and M7. By contrast, ESRD tissue samples that produced faint bands of mRNA produced large amounts of immunoreactivity with M7 (27). These data clearly demonstrate that detection of mRNA does not always predict the expected pattern of protein expression. In contrast to both Ricchiuti and Apple (27) and Messner et al. (28), Haller et al. (26) did not detect cTnT at the mRNA or protein level in skeletal muscle, but did find increased cTnT in the plasma of patients with ESRD. This finding suggests that the plasma cTnT did not originate from skeletal muscle (26). We have concluded that the clearest approach to studying cTnT expression in skeletal muscle in relation to circulating concentrations would be to measure cTnT protein in muscle and in circulation.
Ricchiuti et al. (7) reported positive immunoreactive bands
with M11.7 in <45% of the samples measured and found bands in <4.5%
with M7. In another study using a smaller sample number (n = 7),
the same group found staining in 57% with M11-7 and 29% with M7
(27). These results were not compared with serum cTnT or
confirmed by another technique. The authors loaded into each SDS-PAGE
well 50 µg of protein for both heart extracts and skeletal muscle
extracts, whereas we loaded 2.5 and 15 µg for heart and skeletal
muscle, respectively. Although use of smaller amounts of protein may
increase the limit of detection, we considered it inappropriate to use
equivalent amounts of the two tissues because heart contains several
thousand times more cTnT than skeletal muscle. Because
cross-contamination is a common problem with SDS-PAGE, we minimized the
amount of heart protein to the amount that would produce a positive
signal. Ricchiuti and Apple (27) loaded 50 µg of total
protein for both heart and skeletal muscle extracts and showed a blot
for M7 with the equivalent band intensity for both skeletal and heart
muscle. Although Western blotting is not a quantitative technique,
these findings would suggest that the amounts of cTnT in skeletal and
cardiac muscle are similar, which is in conflict with the final
conclusions drawn in that report, that cTnT is expressed in small
amounts in skeletal muscle. This discrepancy is also illustrated in the
earlier report (7) in which virtually no cTnT was detected
in extracts from cardiac tissue lanes 1 and 2 of Fig. 1
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(7). A large band appeared for skeletal muscle, which was
larger and more intense than those found in heart. Both of these
reports (7)(27) used laser densitometry to
quantify Western blot data.
There is no consensus that muscle weakness associated with CRF is the result of muscle regeneration, and CRF is not a typical condition associated with muscle regression. The mechanism whereby skeletal muscle weakness is associated with CRF is still poorly understood (29), and numerous potential mechanisms have been reviewed recently (30)(31). It has even been suggested that this muscle weakness is not actually caused by myopathy but by neuropathy (9)(32). Only one report with substantive data supports the theory that the muscle weakness sometimes associated with uremia is related to regenerating muscle (12). Diesel et al. (12) compared muscle biopsies from patients with CRF to muscle biopsies from athletes and demonstrated ultrastructural evidence of muscle regeneration (including multinucleated myocytes) in some biopsies from CRF patients (12). In a similar study, however, Bradley et al. (33) found that regenerating fibers were exceedingly rare.
There is a similar controversy surrounding the issue of whether cTnT isoforms are present in myopathic skeletal muscle. Several reports have shown that cTnT is not detected in typically regenerating myopathic muscle. The two most established diseases associated with muscle regeneration are DMD and polymyositis. However, cTnT has been shown not to be abnormally expressed in both of these conditions. Ricchiuti and Apple (27) did not report that cTnT protein was present in skeletal muscle of patients with DMD, as detected by the antibodies M7 and M11-7; in addition, Bodor et al. (15) found quantitatively less cTnT and less CK-MB in skeletal muscle of patients with polymyositis than in healthy adult skeletal muscle. It has also been reported that cTnT was not detected in the skeletal muscle of patients with DMD and the skeletal muscle of the MDX mouse model of DMD by the M7 antibody (34).
Regarding risk stratification among patients with CRF, Porter et al. (35) studied 30 CRF patients over a 1-year period and found that cTnT but not cTnI was predictive, whereas other studies found neither cTnI nor cTnT to be of value in risk stratification (36)(37), and another study found that both markers were strong predictors of risk in patients with normal renal function but that their stratification capacity was reduced in CRF patients (38). A more recent study used a much larger number of patients and studied them over a longer period (102 patients over 2 years) than the previous studies and found a single increased cTnT to be strongly predictive of long-term mortality (39).
The negative results reported here contribute to the discussion of whether the skeletal muscle of patients with ESRD expresses the developmental forms of cardiac proteins. Our finding are in agreement with the conclusions of Ricchiuti and co-workers (7)(27), that cTnT detected in the serum of patients with CRF originates from the heart and not from skeletal muscle. Our findings do not support the theory that CRF patients abnormally express cTnT or CK-MB in skeletal muscle.
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Acknowledgments |
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Footnotes |
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References |
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