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Clinical Chemistry 51: 966-972, 2005. First published April 15, 2005; 10.1373/clinchem.2004.042671
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(Clinical Chemistry. 2005;51:966-972.)
© 2005 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Fast and Slow Skeletal Troponin I in Serum from Patients with Various Skeletal Muscle Disorders: A Pilot Study

Jeremy A. Simpson1, Ralf Labugger1, Christine Collier2, Robert J. Brison3, Steve Iscoe1,a and Jennifer E. Van Eyk1,2

Departments of1 Physiology, 2 Pathology, and 3 Emergency Medicine, Queen’s University, Kingston, Ontario, Canada.

aAddress correspondence to this author at: Department of Physiology, Queen’s University, Kingston, Ontario, Canada K7L 3N6. Fax 613-533-6880; e-mail iscoes{at}post.queensu.ca.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Detection of skeletal muscle injury is hampered by a lack of commercially available assays for serum markers specific for skeletal muscle; serum concentrations of skeletal troponin I (sTnI) could meet this need. Moreover, because sTnI exists in 2 isoforms, slow (ssTnI) and fast (fsTnI), corresponding to slow- and fast-twitch muscles, respectively, it could provide insight into differential injury/recovery of specific fiber types. The purpose of this study was to investigate whether the 2 isoforms of sTnI and their modified forms are present in the blood of patients with various skeletal muscle disorders.

Methods: Serial serum samples were obtained from 25 patients with various skeletal muscle injuries. Serum proteins were separated by a modified sodium dodecyl sulfate–polyacrylamide gel electrophoresis protocol followed by Western blotting for sTnI with monoclonal antibodies specific to ssTnI and fsTnI.

Results: We observed (a) intact and, in some cases, degraded sTnI products; (b) evidence of posttranslational modifications in addition to proteolysis; and (c) differential detectability of both skeletal isoforms in the same patient.

Conclusions: It is possible to monitor both sTnI isoforms; this could lead to the development of new diagnostic assays for skeletal muscle damage.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Currently, the diagnosis of skeletal muscle injury (encompassing all types of muscle diseases) in a clinical setting is a challenge. Direct methods are either invasive (biopsy) or difficult to interpret (e.g., magnetic resonance imaging) (1). Indirect indices of skeletal muscle damage include quantifying muscle soreness, measuring the force generated during maximal voluntary contractions, and monitoring the concentrations of serum markers (1). Despite their widespread experimental use, assessment of muscle soreness and measurement of force during maximal voluntary contraction are not ideal in a clinical setting: the former is subjective and the latter requires a baseline measurement for comparison. Their utility is further compromised by patients who cannot cooperate.

The commonly used serum markers, e.g., lactate dehydrogenase, creatine kinase (CK), 1 and myoglobin, lack specificity to skeletal tissue. Recently, several groups (2)(3)(4)(5) used immunoassays to investigate use of the skeletal isoform of troponin I (sTnI; a myofilament regulatory protein) as a marker of skeletal muscle injury, just as its cardiac isoform (cTnI) is currently the gold standard for detecting cardiac muscle injury (6).

sTnI exists as 2 different isoforms, slow sTnI (ssTnI) and fast sTnI (fsTnI), produced in slow- (ST) and fast-twitch (FT) fibers, respectively. This makes detection of injury to skeletal muscle problematic because human and animal models of injury indicate that it can be FT- or ST-fiber dominant (7)(8)(9)(10)(11)(12)(13)(14). The ability to identify and track injury to specific fiber types is therefore desirable and could provide clinically relevant information.

The usefulness of sTnI assays depends on the ability of the monoclonal antibody (mAb) to detect intact ssTnI and fsTnI and any of their modified forms. Enzymatic (e.g., phosphorylation and degradation) or chemical (e.g., oxidation and acetylation) modifications, collectively referred to as posttranslational modifications (PTMs), of an analyte either before its release or once in the blood can affect antibody binding and thus detection (15). We previously reported (16) that sTnI is proteolyzed in the hypoxemic canine diaphragm. In addition, proteolytic fragments of fsTnI were present in the serum of a patient with drug-induced rhabdomyolysis (17). The development of a sensitive and specific diagnostic assay for sTnI requires characterization of the exact isoforms and form(s), intact or modified, in the blood after injury. The use of Western blot–direct serum analysis (WB-DSA) allows detection of TnI isoform degradation products because it separates reduced and denatured proteins by size, allowing analysis of degraded products and other modifications that could alter mass.

In this pilot study, we used only isoform-specific mAbs for Western blot analysis, allowing simultaneous and independent detection of isoforms and degradation products, to probe for fsTnI and ssTnI in the sera of patients with various skeletal muscle disorders.


   Materials and Methods
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 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patient samples
After obtaining approval from the Research Ethics Board of Queen’s University, we analyzed serum samples from 25 patients admitted to Kingston General Hospital with increased CK concentrations and indications of skeletal muscle disorders. Serum aliquots were obtained from blood samples taken for routine care and not by a defined study time course. Blood was collected in serum separator tubes, centrifuged, subjected to routine biochemical tests, including total CK (Modular Analytics E170; Roche Diagnostics GmbH; upper reference limits of 197 U/L for men and 155 U/L for women), and stored at 4 °C. Aliquots were obtained and subjected to WB-DSA (18). Because this was a pilot study, only limited patient information was available (e.g., onset of disorder, whether acute or chronic, reason for presentation to hospital). Chart review by a clinician was used to classify patients according to their skeletal muscle disorder(s) (Table 1 ).


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  		Table 1. Detection of fast and slow sTnI (intact form and degradation fragments) in patients with skeletal muscle disorders.

serum analysis
Because signal loss has been observed with samples frozen for longer than 6 months, we analyzed samples stored at 4 °C within 10 days of collection. Serum proteins were separated by a modified sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) protocol (18). In brief, SDS-PAGE was performed under denaturing and reducing conditions with a sample buffer containing 3.3 g/L SDS, 3.3 g/L CHAPS, 3.3 g/L NP-40, 0.1 mol/L dithiothreitol, 1.4 mol/L urea, and 50 mmol/L Tris-HCl (pH 6.8) in 100 mL/L glycerol. Serum was diluted 11-fold in sample buffer to prevent precipitation of serum proteins during boiling. Diluted samples were then boiled for 10 min to assure separation of the troponins from serum proteins and to break up binary and ternary complexes. After boiling, 10 µL of diluted serum (equivalent to 1 µL of undiluted serum) was loaded on 12.5% gels and run at 100 V until the dye front reached the bottom of the gel (~3 h).

Gel-electrophoresed proteins were transferred to nitrocellulose (45 µm; Micton Separation, Inc.) in the presence of 10 mmol/L CAPS (pH 11.0) for 45 min at 100 V and 4 °C in a Trans-Blot Cell apparatus (Bio-Rad). Nitrocellulose blots were transiently stained with Ponceau S (Sigma) to identify albumin, which nonspecifically binds both primary and secondary antibodies. Blots were then cut at ~50 kDa to remove the albumin band. Membranes were blocked overnight at 4 °C in 100 mL/L blocking reagent (Roche). Primary antibodies of confirmed isoform specificity were used (16)(17). These included FI-32 and FI-23 (Spectral Diagnostics) and SI-1 (Hytest), which are specific for fsTnI, and MYNT-S (courtesy of N. Matsumoto), which is specific for ssTnI. At the time of this work, these mAbs were the best available, based on our evaluation of 27 different anti-sTnI mAbs. Blots were incubated with primary antibody (0.5 mg/L) followed by an anti-mouse IgG antibody–alkaline phosphatase conjugate (Jackson Laboratories). All antibodies were diluted in 10 mL/L blocking reagent and incubated for 45 min at room temperature. Signals were visualized with chemiluminescence substrate (Roche) and X-Omat Scientific Imaging Film (Eastman Kodak). Exposure times of Western blots were chosen to maximize visualization of intact sTnI and/or its degradation products; consequently, comparisons of the intensities of bands from different patients were not possible.

A sample was considered positive when ≥2 mAbs detected the isoform and/or its fragments and the intact isoform migrated at the appropriate molecular mass. Serum known to be troponin negative was used as a control. Serial dilution studies of serum supplemented with fsTnI indicated that we can detect down to at least ~1.56 pg per lane (data not shown).


   Results
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 Materials and Methods
 Results
Discussion
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fsTnI was detected in the blood of all 25 patients and ssTnI in 16 of the 21 patients tested (Table 1Up ). Representative Western blots of serum samples from 5 different patients, probed independently for fsTnI and ssTnI, are shown in Fig. 1A . The lower band of the doublet of fsTnI migrated with intact fsTnI, as determined from serum samples supplemented with skeletal muscle tissue (data not shown). Both slow and fast isoforms were detected, and both had degradation products, although not in all patients. On the basis of migration on 1-dimensional SDS-PAGE, a maximum of 7 proteolytic fragments were observed for fsTnI and 3 for ssTnI. The extent of proteolysis and number of fragments varied among patients, but this was unrelated to CK concentrations (compare the blots of patients A and B with their corresponding CK concentrations; Fig. 1B ). Proteolytic fragments of fsTnI and ssTnI were observed in only 13 of 25 (52%) and 7 of 16 (44%) patients, respectively (Table 1Up ).



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  		Figure 1. sTnI detection in serum of patients with various skeletal muscle disorders.

(A), representative Western blots of serial serum samples from 5 of the 25 patients (first blood sample on hospital admission is considered time 0), probed for fsTnI (top) and ssTnI (bottom). Both isoforms, as well as several modified forms (proteolytic fragments of fsTnI and ssTnI as well as a doublet of only the intact fsTnI isoform), were detected. (B), the presence and number of proteolytic fragments varied among patients and were unrelated to CK concentrations. The upper row of CK values below the gels represent those of the serum samples used for WB-DSA, the lower row represent the peak values during hospitalization, typically in samples taken within 6 h of the sample taken for WB-DSA.

The temporal immunoreactivity profiles obtained with 2 fsTnI-specific mAbs in 8 samples from 2 different patients are shown in Fig. 2 . In the first patient (Fig. 2A ), mAb SI-1 (bottom panel) displayed less immunoreactivity in the first 4 samples than in the last 4, whereas mAb FI-32 (top panel) displayed relatively equal immunoreactivities in all 8 samples. The differential detection of fsTnI and ssTnI over time in another patient is shown in Fig. 2B . The initial increase in ssTnI was followed by a rapid decrease to lower concentrations (prolonged exposures were required for visualization). In contrast, fsTnI increased, peaking after ssTnI peaked, and remained increased.



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  		Figure 2. Differential detection of the isoforms and modified forms of sTnI.

(A), serial serum samples from patient 14, probed for fsTnI with 2 different mAbs. Although mAb FI-32 detected approximately equal concentrations of fsTnI over the 3 days, mAb SI-1 showed a pronounced increase in immunoreactivity during the course of the disease. (B), Serial serum samples from patient 1, probed for fsTnI and ssTnI.

Although both isoforms were detected in most patients (Table 1Up , patients 1–4, 6–11, and 13–18), only the fast isoform was detected in the sera of others (patients 20–23 and 25) with activity-induced (exercise or exertion) injury. The findings in 2 such patients are shown in Fig. 3 . One patient had both cardiac [confirmed by cardiac troponin T (cTnT)] and respiratory complications (Fig. 3A ), whereas the other had only respiratory complications (Fig. 3B ; no increased cTnT).



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  		Figure 3. Only fast sTnI was detected in some patients.

(A), patient 22, who presented with both cardiac and respiratory complications. (B), patient 21, who presented with only respiratory complications. ssTnI was not detected.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using WB-DSA, we have detected both slow and fast isoforms of sTnI in patients with skeletal muscle disorders, raising the possibility that their detection may eventually provide useful diagnostic information. A limitation of our technique, WB-DSA, is that it cannot detect PTMs (e.g., oxidation and glycosylation) that do not alter the electrophoretic migration of the protein on 1-dimensional SDS-PAGE. However, differential detection of the same isoform by different mAbs indicates the presence of a PTM (Fig. 2Up ).

The doublet of the intact fsTnI isoform (Fig. 1AUp ) is not the result of proteolysis but a different modification. Phosphorylation is one type of modification that can cause a shift in electrophoretic mobility on 1-dimensional SDS-PAGE, creating a doublet, the parent protein with the higher molecular mass band (~2–4 kDa) being the phosphorylated form (19)(20). Although we found no difference in the ratio of the 2 intact fsTnI bands after dephosphorylation with alkaline phosphatase (18), this result does not exclude the possibility that the upper band was a phosphorylated form (or some other PTM, e.g., oxidation, glycosylation, or nitration) of fsTnI. Of interest, the ratios of the 2 bands varied among patients (Fig. 1BUp ) independent of proteolysis and/or CK concentrations. Both of these results, the doublet and variations in its ratio, are similar to those observed for cTnI in the sera of patients after acute myocardial infarction (18).

The nature of modifications changed during the course of the disease (Fig. 2Up ). The altered immunoreactivity of mAb SI-1 does not reflect changes in analyte concentration because mAb FI-32 revealed relatively constant amounts throughout. Thus, differential detection of fsTnI by mAbs FI-32 and SI-1 indicates the presence of a modified form that affects the binding of mAb SI-1 but not FI-32. The clinical relevance of these modifications remains to be determined.

Did proteolysis occur in the tissue or after its release into blood? We previously showed, in severely hypoxemic dogs, that proteolysis of sTnI occurs in the diaphragm, demonstrating that proteolytic fragments form in the muscle cell before release (16). This is similar to what happens in the myocardium, where the cTnI undergoes specific and progressive proteolysis (21)(22); recombinant human cTnI added to serum undergoes little, if any, proteolysis over 24 h at 37 °C (18). Considering the biochemical similarities of cardiac and skeletal TnI, these results suggest that proteolysis of sTnI occurs in the tissue rather than after release.

The ability to monitor independently both sTnI isoforms permits tracking of injury to each fiber type. Skeletal muscles are generally classified as either ST (also referred to as type I) or FT (also referred to as type IIA and IIB) based on histochemical staining for myosin adenosine triphosphatase [for a review, see Ref. (23)]. ST fibers are better equipped to work aerobically, whereas FT fibers are better equipped to work anaerobically. Most adult human muscles are composed of various proportions of ST and FT fibers, allowing them to function over a wide range of contractile demands.

Several studies in animals and humans have suggested that activity-induced injury can be FT- or ST-fiber dominant, but this issue is unresolved. Eccentric or concentric contraction-induced injury may be preferential for FT (8)(9)(10)(14) or ST (10)(12)(13)(14) fibers. Vijayan et al. (11) proposed that selective recruitment of fiber types explains, in part, fiber-specific injury. In addition, the biopsy procedure itself can produce damage, mimicking exercise-induced injury (24). Moreover, because ultrastructural injury is generally patchy, involving a small percentage of cells, the limited amount of tissue sampled (compared with that of the whole muscle) may lead to over- or underestimation of the extent of injury. In addition, histologic techniques are believed to be too insensitive to detect low-level cellular injury (25).

Unlike exercise, ischemia–reperfusion causes similar degrees of injury in FT and ST fibers (26). Targeting of isoform-specific proteins may overcome the problems associated with classic methods of detecting fiber-type–specific damage. Our finding that only fsTnI was detected in the blood of patients presenting with activity-induced injury is identical to that reported recently in rats with diaphragmatic fatigue and respiratory failure caused by breathing against an inspiratory resistive load (27). These results suggest that activity injures only FT fibers.

In our patients, we observed no relationships between CK concentrations, either mean or peak, and the extent or nature of proteolysis (Fig. 1BUp ). If CK concentrations indeed reflect the amount of injured muscle (28), then proteolysis may be related not just to the amount of tissue damage, but also (or rather) to the nature of the stress to which the muscle cells are exposed.

Another issue related to reliance on CK is that its tissue of origin may not always be readily apparent. Skeletal muscle injury may not be diagnosed when traditional serum markers such as CK are used because of "contamination" of the skeletal signal by cardiac injury (e.g., see Fig. 3AUp ). Thus, one could wrongly infer that the increase in CK was a result of the myocardial infarction (increased cTnT) rather than skeletal muscle injury.

To our knowledge, only 2 companies have tried to develop a skeletal muscle assay using sTnI as a biomarker, but neither assay could differentiate between skeletal and cardiac muscle injury because of extensive cross-reactivity of the mAbs with the cardiac isoform. Furthermore, these assays could not differentiate between the 2 skeletal isoforms. Takahashi et al. (5) and Sorichter et al. (2) used these assays to investigate skeletal muscle injury/disease. In both studies, the focus was on acute injuries, but Takahashi et al. (5) also investigated serum sTnI concentrations in patients with Duchenne muscular dystrophy and polymyositis, both examples of progressive degenerative muscle diseases. However, both studies were limited because fiber-specific injury cannot be tracked. In addition, although both of the aforementioned assays possess analytical sensitivity, their diagnostic sensitivity could have been compromised by PTMs affecting the antibody affinity of sTnI, as shown in this study (Fig. 2AUp ).

In conclusion, skeletal muscle injury led to the release of one or both isoforms of sTnI and their modified products into serum; activity-induced injury preferentially injured FT fibers. Because sTnI, unlike cTnI, can be released from any skeletal muscle, its source cannot be determined, but a careful history should reveal the likely source(s). Detection of different forms and isoforms of sTnI raises the possibility that we may be able to determine not just the origin but also the nature of the injury and its severity. Our results emphasize the need to develop an assay that can exploit the potential of sTnI as a diagnostic marker.


   Acknowledgments
 
J.A.S. was supported by a studentship from the government of Ontario, funds allocated to Queen’s University from the Ontario Thoracic Society, the Wm. M. Spear Foundation, and the School of Graduate Studies and Research, Queen’s University. This work was supported by grants to S.I. and J.V.E. from the Canadian Institutes of Health Research (MOP 36339, 64367, and 14375), the Ontario Thoracic Society, and the Heart and Stroke Foundation of Canada (T-3759). J.V.E. received salary support from the Hopkins NHLBI Proteomics Grant, Contract NO-HV-28180, and the DW Reynolds Foundation. We thank Spectral Diagnostics Inc. (Toronto, Canada) and Dr. N. Matsumoto (Department of Internal Medicine, Nanasawa Rehabilitation Hospital, Kanagawa, Japan) for generously providing antibodies for this study.


   Footnotes
 
2 Current address: 602 Mason F. Lord Building, Center Tower, Johns Hopkins University, Baltimore, MD 21224.

1 Nonstandard abbreviations: CK, creatine kinase; fsTnI and ssTnI, fast and slow skeletal troponin I, respectively; cTnI and cTnT, cardiac troponin I and T, respectively; ST and FT, slow and fast twitch, respectively; mAb, monoclonal antibody; PTM, posttranslational modification; WB-DSA, Western blot–direct serum analysis; and SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
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 References
 

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