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Department of Laboratory Medicine and Pathology, Hennepin County Medical Center, University of Minnesota School of Medicine, Minneapolis, MN 55415.
a Address correspondence to this author at: Hennepin County Medical Center, Clinical Laboratories, Mail Code 812, 701 Park Avenue South, Minneapolis, MN 55415. Fax 612-904-4229; e-mail fred.apple{at}co.hennepin.mn.us
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Total mRNA was extracted from healthy human heart (n = 4), healthy human skeletal muscle (n = 5), and skeletal muscle from patients with ESRD (n = 7) and DMD (n = 5). Total RNA (1 µg) was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase. The reverse-transcribed cDNAs were amplified by PCR using oligonucleotide primers specific for cTnT, sTnT, and cTnI sequences (GenBank accession numbers X74819, m19308, and X54163, respectively).
Results: In all heart specimens, a 150-bp cTnT amplicon was detected. Skeletal muscle from four of seven patients with ESRD and two of five patients with DMD showed expression of a 150-bp amplicon. Using DNA sequencing and a comparison program, the 150-bp amplicons found in heart and diseased skeletal muscle specimens were 100% identical and specific to the cTnT mRNA sequence. No cTnT mRNA expression was found in healthy skeletal muscle. No evidence of sTnT mRNA was found in heart muscle. A 200-bp sTnT amplicon specific to a human sTnT sequence was detected in all skeletal muscle specimens. A 250-bp cTnI amplicon specific to the cTnI sequence was detected in all heart specimens. However, no cTnI mRNA expression was found in healthy or diseased skeletal muscle specimens. cTnT mRNA expression in both heart and diseased skeletal muscles corresponded with cTnT isoform expression, respectively, as determined by Western blot analysis.
Conclusion: Our findings demonstrate cTnT mRNA expression, but no cTnI mRNA expression, by reverse transcription-PCR in diseased human skeletal muscle that expresses cTnT isoforms.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies using immunochemical staining analysis and Western blot analysis have demonstrated that several cTnT isoforms are re-expressed during regeneration in adult rat skeletal muscle after injury or denervation (6) and in human skeletal muscle from patients with Duchenne muscular dystrophy (DMD) (7), polymyositis (7), and end-stage renal disease (ESRD) (8). The mechanism for expression of cTnT isoforms in skeletal muscle from ESRD patients is likely associated with peripheral myopathy associated with renal disease (9). Expression of cTnT isoforms in diseased or regenerating skeletal muscle appears to represent re-expression of the cTnT gene, given that fetal skeletal muscle expresses cTnT (6).
The presence of circulating cTnT and cTnI in blood is a specific indicator of heart muscle damage. This observation serves as the basis for using cTnT and cTnI measurement to diagnose acute myocardial infarction (10)(11). Furthermore, the observations that cTnT and cTnI can be released during unstable angina led to the recognition of small amounts of myocardial damage (12)(13)(14). The incidence and prognostic value of increased cTnT and cTnI concentrations in chronic hemodialysis patients, independent of their history of coronary artery disease, has not been fully characterized. In a previous study, however, we carefully characterized cTnT isoform expression in skeletal muscle from patients with ESRD using the two MAbs from the second- and third-generation Roche cTnT immunoassays and demonstrated that measurable cTnT in the circulation is cardiac specific (15).
The purpose of the current study was to determine mRNA and protein expression of cTnT and cTnI in diseased and healthy human skeletal muscle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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rna isolation from patient muscles
RNA was extracted from all heart and skeletal muscle tissues.
Fifty to 100 mg of each tissue was homogenized with a Polytron
homogenizer (Brinkman Instruments), using a commercial procedure
modified from the guanidine-isothiocyanate-phenol method developed by
Chomczynski and Sacchi (16). The cell components were
disrupted in 1 mL of TRIzol reagent (Life
TechnologiesTM), and homogenates were
incubated at room temperature for 30 min. After the addition of 0.2 mL
of chloroform per 1 mL of homogenate, tubes were mixed by vortex-mixing
for 5 s and incubated at room temperature for 5 min. Samples
were centrifuged at 12 000g for 15 min at 4 °C. RNA was
recovered from the upper aqueous phase (600 µL) by precipitation with
0.5 mL of isopropyl alcohol. Samples were incubated at room temperature
for 15 min and centrifuged at 12 000g for 15 min at
4 °C. The supernatants were removed, and the RNA pellets were washed
twice with 1 mL of 750 mL/L ethanol. The samples were mixed by
vortex-mixing and centrifuged at 7500g for 5 min at 4 °C.
The RNA pellets were air dried 5 min at room temperature and
reconstituted in 20 µL of RNase-free water. The purity of the RNA was
determined from the ratio of absorbance readings at 260 and 280 nm,
with an A260/280 ratio between 1.8 and
2.0 indicating sufficient purity. The concentration of RNA was
determined from the absorbance at 260 nm. RNA samples were kept frozen
at -75 °C until used.
reverse transcription-pcr (rt-pcr) of troponin in skeletal muscle
One microgram of total RNA was reverse-transcribed using Moloney
murine leukemia virus reverse transcriptase
(SuperScriptTM II; Life Technologies) according
to the supplier’s protocol. Forward and reverse primers were designed
using the primer design and analysis software Oligo 6.01 (Molecular
Biology Insights). The reverse-transcribed cDNAs were amplified by PCR
using the following oligonucleotide primers:
(a) Human cTnT amplification (17): Forward: 5'-GGCAGCGGAAGAGGATGCTGAA-3' Reverse: 5'-GAGGCACCAAGTTGGGCATGAACGA-3'
(b) Human slow skeletal muscle troponin T (sTnT) amplification (18): Forward: 5'-GCGGCTACCTGGTCAAGGCAGAA-3' Reverse: 5'-GAGGCACCAAGTTGGGCATGAACGA-3'
(c) Human cTnI amplification (19): Forward: 5'-CCCTGCACCAGCCCCAATCAGA-3' Reverse: 5'-CGAAGCCCAGCCCGGTCAACT-3'
The following components were combined in PCR reaction tubes: 5 µL of 10x PCR buffer solution (200 mmol/L Tris-HCl, pH 8.4, 500 mmol/L KCl; Promega); 3 µL of 25 mmol/L MgCl2 solution (GenAmp® PCR products); 1 µL of dNTP solution (10 mmol/L); 0.5 µL of Taq Polymerase solution (5 U/µL; Promega); 2 µL of forward primer and reverse primer solutions, both at 15 µmol/L; 2 µL of reverse-transcribed cDNAs; and 36.5 µL of autoclaved distilled water with a final reaction volume of 50 µL. Samples were mixed gently, and 2 drops of silicon oil (Sigma) were layered over the reaction solution. The reaction tubes were heated at 94 °C for 3 min for denaturation, followed by 40 cycles of PCR at 94 °C for 10 s and 68 °C for 45 s in a DNA thermal Cycler (GenAmp PCR System 2400; Perkin-Elmer). The amplified DNA fragments were visualized by 4% modified agarose gel (NuSieve®; FMC BioProducts) electrophoresis combined with ethidium bromide staining. Two micrograms of 100-bp DNA ladder (100–2000 bp; Life Technologies) was used as a reference control to estimate the PCR amplicon length. The amplicons obtained by RT-PCR from heart and skeletal muscle mRNAs were compared using DNA sequencing (University of Maine System, Orono, ME) and a sequence comparison software program (Genetic Computer Group, Ver. 9.1).
protein extraction
As described previously (15), all samples (~50 mg) of
frozen nondiseased human heart muscle (n = 5), nondiseased human
skeletal muscle (n = 5), and diseased skeletal muscle from
patients with ESRD (n = 7) or DMD (n = 5) were coarsely
ground in a liquid nitrogen-cooled mortar and then added to a protein
extraction buffer (200 mmol/L potassium phosphate, pH 7.4, 5.0 mmol/L
EGTA, 5.0 mmol/L ß-mercaptoethanol, and 100 mL/L glycerol). The
samples were homogenized at 4 °C. The supernatants were used
immediately for protein analysis and Western blotting.
antibodies
Three different primary MAbs were selected for use in Western
blotting, as described previously (15). A mouse MAb specific
for cTnI (JS-1; specific residues recognized on cTnI protein sequence
not available from manufacturer) was a gift from Lakeland
Biomedical, Minneapolis, MN, and was used at 2 mg/L (7). Two
MAbs specific for cTnT were used (both at a 2 mg/L). MAbs M7 and M11.7,
which recognize residues 125–131 and 136–147, respectively, of the
cTnT protein sequence, were provided by Dr. Klaus Hallermayer, Roche
Diagnostics, Tutzing, Germany (20). MAbs M11.7 and M7 are
the capture and detection antibodies in the Roche cTnT second- and
third-generation immunoassays.
western blot analysis
As described previously (15), 50-µg samples of all
protein extracts were size-fractionated on sodium dodecyl
sulfate-polyacrylamide gels using the method of Laemmli (21)
with the following modifications: 30% acrylamide and 1.1%
bis-acrylamide stock solutions were used in 7.5% running gels and
3.3% stacking gels (4). Proteins were subsequently
transferred to nitrocellulose membrane. After the blocking step, the
primary antibody, as described above, was diluted in antibody buffer
and incubated with the membrane. The membranes were then washed three
times. Appropriate horseradish peroxidase-labeled secondary antibodies
(sheep anti-mouse) were then incubated with the membranes for 1 h.
The membranes were again washed three times before a 1-min incubation
with ECLTM chemiluminescent substrate (Amersham).
Light emission was detected by exposure to Fuji RX autoradiography film
in the presence of Cronex intensifying screens (Fisher Scientific).
Signal intensities within the linear range were quantified by laser
densitometry (Molecular Dynamics).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the RT-PCR amplification of cTnT (Fig. 1A
), cTnI (Fig. 1B
) and sTnT (Fig. 1C
) cDNA sequences from reverse-transcribed heart
and skeletal muscle RNAs. PCR amplification was successful with cardiac
muscle-specific primers for cTnT (Fig. 1A
) and cTnI (Fig. 1B
) when used
on heart muscle total RNA (lanes 1–3). No PCR amplification of cardiac
primers in reverse-transcribed RNA was found in healthy skeletal muscle
samples (cTnT, lanes 4–6 in Fig. 1A
; cTnI, lanes 4–6 in Fig. 1B
).
Four of seven skeletal muscle specimens from patients with ESRD (lanes
7, 9, 10, and 12) and two of five skeletal muscle specimens from
patients with DMD (lanes 14 and 15) showed amplification at the
expected length (150 bp) for cTnT (Fig. 1A
) but not for cTnI (Fig. 1B
).
We controlled for the presence of intact sTnT mRNA in each biopsy
sample tested using primers specific for slow twitch skeletal muscle
sequence. No evidence of sTnT mRNA was found in any of the biopsies
from healthy hearts (Fig. 1C
, lanes 1–3). However, a 200-bp sTnT
amplicon was detected in all biopsies from skeletal muscle specimens
(Fig. 1C
, lanes 4–15).
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DNA sequencing analysis for the most representative cTnT amplicons
obtained by RT-PCR is shown in Fig. 2
. The comparison analysis of these amplicons with sequences from
GenBank database showed that the cTnT 150-bp amplicons found in heart
and diseased skeletal muscle specimens were 100% identical and
specific to the human cTnT cDNA sequence (GenBank accession number
X74819). The cTnI 250-bp amplicons found in heart specimens were 100%
identical and specific to the human cTnI cDNA sequence (GenBank
accession number X54163; data not shown). The sTnT 200-bp amplicons
found in healthy and diseased skeletal muscle specimens were 100%
identical and specific to the human slow twitch skeletal TnT
cDNA sequence (GenBank accession number m19308; data not shown).
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Figs. 3
and
4 show that heart muscle samples demonstrated both mRNA and
protein expression for cTnT (250-bp amplicon product and one major
39-kDa isoform and one minor 34-kDa isoform) and for cTnI
(150-bp amplicon product and one major 25-kDa isoform). None of the
healthy skeletal muscle samples expressed either cTnT or cTnI protein
or RNA (Figs. 3
and 4
). However, the four ESRD skeletal muscle samples
that demonstrated cTnT isoforms at 34–36 kDa, as well as the two ESRD
muscles that demonstrated a 39-kDa cTnT isoform also showed expression
of cTnT RNA by a single amplicon product (Fig. 3
). No expression of
either cTnI protein or mRNA was demonstrated in any diseased skeletal
muscles (Fig. 4
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). cTnT
primers were designed from the cDNA sequence with the human genomic
sequence (GenBank accession number X74819). Therefore, the primers used
in this study are parts of exons. We controlled for the possibility of
contaminating genomic DNA by running PCR on RNA extraction specimens
with different sets of primers (cTnT-specific and actin-specific). None
of the RNA extraction samples showed any presence of contaminating
genomic DNA. The current findings contrast with the recent report by Haller et al. (22), which showed that no evidence of cTnT expression at the mRNA or protein level was demonstrated in truncal skeletal muscle biopsies from five patients with ESRD. One of the limitations in the study by Haller et al. (22) was that the anti-cTnT antibody used had reportedly cross-reacted with human skeletal muscle troponin T isoforms. The reduced specificity of the antibody and the low resolution of the immunoblot analysis shown in their report may have limited their conclusions regarding cTnT expression in the skeletal muscle of dialysis patients. Another limitation of the study by Haller et al. (22) was that both immunoblot and PCR analysis involved only four biopsies from patients with an increased serum concentration of cTnT. However, our previous study involving 45 biopsy extracts from patients with ESRD screened with specific anti-cTnT antibodies revealed that <50% will show expression of cTnT isoforms within the range 34–36 kDa (MAb M11.7) and that <5% will show expression of 39-kDa cTnT isoforms (MAb M7) (15).
The main findings in the current study were obtained using RT-PCR analysis and oligonucleotide primers specific for the cTnT and cTnI isoforms and the skeletal muscle isoform of troponin T. We showed isoform-specific amplification products in both cardiac and skeletal muscle tissues. We demonstrated the RT-PCR amplification of cTnT, cTnI, and sTnT cDNA sequences from reverse-transcribed muscle RNA. PCR amplification was successful with cardiac muscle-specific primers when used on heart muscle total RNA. No PCR amplification of cardiac primers in reverse-transcribed RNA was found for healthy skeletal muscle samples. Four of seven skeletal muscle specimens from patients with ESRD and two of five skeletal muscle specimens from patients with DMD showed amplification at the expected length (150 bp) for cTnT but not for cTnI. We controlled for the presence of intact sTnT mRNA in each biopsy sample by using primers specific to the slow twitch skeletal muscle sequence. No evidence of sTnT mRNA was found in any of the heart biopsies. However, a 200-bp sTnT amplicon was detected in all skeletal muscle specimens. These results show that all the skeletal muscle specimens, including the ones that did not amplify any cTnT cDNAs in PCR, contained intact cDNAs after reverse transcription and, therefore, contained intact total mRNA.
When we compared our current findings with our previously published results regarding cTnT protein expression using anti-cTnT M11.7 and M7 antibodies (15) with RNA expression by RT-PCR analysis in the same biopsy specimens, we found excellent agreement. MAbs M11.7 and M7 are designated as capture and detection antibodies, respectively, in the Roche cTnT second- and third-generation immunoassays. In all specimens from the heart, one major cTnT isoform, with a molecular mass of 39 kDa, and one minor cTnT isoform, with a molecular mass of 34 kDa, were recognized by MAbs M11.7 and M7. These same specimens demonstrated RNA expression of cTnT by a single amplicon product (150 bp). None of the five specimens from healthy skeletal muscle showed either protein or RNA expression of cTnT. Four of seven specimens that demonstrated expression of cTnT protein isoforms (34–36 kDa) using MAb M11.7 also showed expression of cTnT mRNA by a single amplicon product. The different protein isoforms detected by M11.7 appear to reveal potentially important posttranscriptional (splicing) differences between different ESRD muscle tissues. Both specimens that demonstrated an expression of the 39-kDa cTnT protein isoform using MAb M7 showed expression of cTnT mRNA. Furthermore, as in our previous study (15), we again demonstrated that the anti-cTnT M11.7 and M7 Roche antibodies recognized different epitopes on cTnT isoforms expressed in diseased skeletal muscle. Therefore, even if these cTnT isoforms were released from skeletal muscle into the circulation, they would not be measured by the Roche second- and third-generation cTnT immunoassays.
Our current findings (Fig. 4
) also concur with our previous findings
(15), demonstrating that one major cTnI isoform, with a
molecular mass of 25 kDa, was recognized by MAb anti-cTnI JS-1 in heart
specimens. This corresponded to mRNA expression of cTnI by a single
amplicon product (200 bp, using identical heart specimens). No
expression of cTnI protein or mRNA was demonstrated in any of the
healthy or diseased skeletal muscle specimens. Furthermore, the 200-bp
amplicons found in healthy and diseased skeletal muscle specimens were
100% identical and specific to the human slow twitch skeletal
TnT mRNA sequence. The absence of extracardiac cTnI expression
in diseased skeletal muscle lends additional support to the hypothesis
that the cTnI found in the serum of ESRD or DMD patients originates
only from the heart (23).
In human cardiac muscle, multiple isoforms of cTnT have been described, which are expressed in fetal, adult, and diseased heart, resulting from alternative splicing of a single cTnT gene (3)(24)(25) composed of 17 exons spread over 17 kb. A potential structure of the promoter region has been proposed, and several polymorphisms in both the exonic and intronic regions were identified recently, some of which may act as modulators of the expression of the cTnT gene (26).
However, the precise physiological relevance of the TnT isoforms in human heart currently is poorly understood. Recently, the cTnT gene was located at the CMH2 locus on chromosome 1q32 (17)(25), and mutations in its sequence have been found to be associated with familial hypertropic cardiomyopathy (27)(28). The re-expression of multiple isoforms of cTnT in diseased human skeletal muscle parallels, and probably results from, the expression of these isoforms in differentiating myotubes (29) and is consistent with the expression of developmentally expressed fetal isoforms, as described previously for both cTnT (5)(24) and creatine kinase isoenzymes (30).
Previous studies have shown that in patients with ESRD, serum concentrations of both cTnT and cTnI are increased without evidence of cardiac ischemia (8). Other studies have shown that serum concentrations of cTnT are significantly lower in patients with fewer cardiac risk factors compared with patients with known coronary artery disease (22). The clinical implications of the current study provide additional evidence consistent with the hypothesis that circulating cTnT or cTnI in either ESRD or DMD patients originates from the heart.
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Acknowledgments |
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Footnotes |
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References |
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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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M. Beciani, A. Tedesco, A. Violante, S. Cipriani, M. Azzarito, A. Sturniolo, and G. Splendiani Cardiac troponin I (2nd generation assay) in chronic haemodialysis patients: prevalence and prognostic value Nephrol. Dial. Transplant., May 1, 2003; 18(5): 942 - 946. [Abstract] [Full Text] [PDF]
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M. C. Iliou, C. Fumeron, M. O. Benoit, P. Tuppin, C. L. Courvoisier, V. M. Calonge, N. Moatti, C. Buisson, and C. Jacquot Factors associated with increased serum levels of cardiac troponins T and I in chronic haemodialysis patients: Chronic Haemodialysis And New Cardiac Markers Evaluation (CHANCE) study Nephrol. Dial. Transplant., July 1, 2001; 16(7): 1452 - 1458. [Abstract] [Full Text] [PDF]
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