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Technical Briefs |
Departments of
1
Physiology,
2
Pathology,
3
Respirology, and
4
Biochemistry, Queen’s University, and
5
Kingston General Hospital, Kingston, Ontario, K7L 3N6 Canada;
6
Internal Medicine, Sagamihara Kyodo Hospital, Hashimoto 2-8-18, Sagamihara, Kanagawa 229, Japan
aauthor for correspondence: Queen’s University, Department of Physiology, Kingston, Ontario, K7L 3N6 Canada; fax 613-533-6880, e-mail jve1{at}post.queensu.ca
Unlike the extensive research devoted to the development of troponin-based diagnostic assays for myocardial disease, much less effort has been expended on the development of a counterpart for skeletal muscle disorders. The consensus that the cardiac troponins [cardiac troponin I (cTnI) and/or cTnT] should be used for diagnosis of myocardial infarction (MI), as well as for diagnosis and management of unstable angina, is based on their superior tissue specificity over such conventional markers as creatine kinase [(CK); see Refs. (1)(2)]. Although CK is the most common serum marker for skeletal muscle injury, it is not ideal for several reasons, including lack of tissue specificity, inability to reveal damage to specific skeletal fiber types (fast or slow), and inappropriately low values when glutathione concentrations are decreased because of liver or multiple-organ failure (3). Skeletal troponin I (sTnI), with its two distinct isoforms [fast (fsTnI) and slow (ssTnI)], like cTnI and cTnT, may have a similar advantage over conventional markers for detecting skeletal-muscle injury.
In 1996, Rama et al. (4) described an experimental immunoenzymatic assay for sTnI using antibodies that cross-react with both sTnI and cTnI. The assumption of the investigators was that the concentrations of cTnI in patients with skeletal injury would be negligible. Others have since used this assay (5)(6)(7). For example, Onuoha et al. (7) found that serum sTnI reflects the severity and type of orthopedic and soft tissue injury. However, because this assay does not differentiate between the two isoforms of sTnI, which have a sequence homology of 
56%, information about selective damage to particular fiber types is unavailable. Posttranslational modifications to the analyte, such as degradation, are also undetected by this assay.
We applied our Western blot–direct serum analysis (WB-DSA) procedure (8), originally developed and successfully used for the detection of cardiac troponins in serum of patients with MI, to investigate the selective detection of serum fsTnI and ssTnI in a case of skeletal muscle disease. Polyacrylamide gel electrophoresis (12%) on 1 µL of undiluted serum was performed under denaturing and reducing conditions, and proteins were transferred to nitrocellulose membranes for Western blot analysis according to a previously described protocol (8). We tested 24 different antibodies for their specificity against fsTnI and ssTnI by Western blot analysis using tissue from several striated muscles, including human myocardium (cTnI only), feline soleus (ssTnI only), feline caudofemoralis (fsTnI only), rat diaphragm (ssTnI and fsTnI), and human diaphragm (ssTnI and fsTnI), as described previously (9). Because rat and human diaphragms are of mixed fiber types and the isoforms have slightly different molecular masses, fsTnI (24 kDa) and ssTnI (25 kDa) appear as two distinct bands on 12% polyacrylamide gels (9). Thus, antibodies either detected both isoforms (two bands by WB-DSA) or one isoform (one band). Isoform specificity was further confirmed by testing the antibodies against muscles of known fiber types [feline soleus, 100% slow; feline caudofemoralis, 100% fast; see Ref. (10)]. The amino acid sequences for feline and swine sTnI, unlike those for human, rat, rabbit, and mouse sTnI, are unknown, but fsTnI and ssTnI show sequence homologies of 95% and 98%, respectively, between mammals. Two antibodies with confirmed isoform specificity (Fig. 1
, C and D) were chosen for WB-DSA: monoclonal anti-human fsTnI antibody F32 (0.5 mg/L; courtesy of Spectral Diagnostics Inc., Toronto, Canada) and monoclonal anti-human ssTnI antibody MYNT-S [0.5 mg/L; see Ref. (11)]. These antibodies, neither of which cross-reacted with cTnI, showed the same isoform specificity in all species tested.
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In a preliminary study, we analyzed serum samples (n = 20; obtained over 8 days) from a patient admitted with drug-induced rhabdomyolysis to Kingston General Hospital (Ontario, Canada). The study was approved by the Human Research Ethics Board of Queen’s University. Serum aliquots were obtained from blood samples taken for routine care, not by a defined study time course. Serum was assayed immediately for routine laboratory testing or frozen for subsequent WB-DSA. Routine testing included total CK (Modular Analytics E170; Roche Diagnostics GmbH; upper reference limits of 197 U/L for men and 155 U/L for women) and cTnT (Elecsys 1010; Roche Diagnostics GmbH; upper reference limit, 0.05 µg/L; Table 1
).
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Selective detection of fsTnI and ssTnI by WB-DSA revealed that both isoforms were present in the patient’s serum and that their release and/or clearance patterns differed over time (Fig. 1
, A and B). The fsTnI decreased by the end of day 6 (sample 15) and increased again until day 8 (sample 20, the last sample taken before the patient died within 24 h). Like fsTnI, ssTnI also decreased by day 6, but remained low. Interestingly, the total CK serum profile (with a rapid decrease starting during day 7) resembled the time profile of ssTnI rather than that of fsTnI (as determined by WB-DSA; compare Table 1
with Fig. 1
, A and B).
One of the main advantages of WB-DSA over other immunoenzymatic assays is the ability to detect not only the presence of the specific isoforms but also some posttranslational modifications. In this patient, fsTnI underwent proteolytic degradation and showed a doublet of the intact protein, comparable to what can be observed for cTnI in patients with MI (8). Because degraded products of ssTnI were detectable only with prolonged exposures, the two isoforms may have different susceptibilities to enzymatic proteolysis and posttranslational modification, either within the muscle or after their release into blood.
In this preliminary study, using WB-DSA, we were able to detect sTnI in serum from a patient with rhabdomyolysis. We do not know whether these changes in fsTnI and/or ssTnI indicated the patient’s condition and could therefore have served as a useful biomarker of clinical status. Unlike other assays, our selected antibodies enabled us to differentiate between the two sTnI isoforms, revealing differences in their release and/or clearance patterns and that both had undergone posttranslational modifications. Skeletal and cardiac TnI isoforms share 60% of their amino acid sequence, and the selection of antibodies therefore determines assay specificity for cardiac or skeletal tissue (12). The advantage of antibodies specific for the skeletal isoforms [unlike those that bind both cTnI and sTnI, as used in the assay of Rama et al. (4)] is that they eliminate the influence of confounding cardiac disease (4)(13)(14)(15)(16), as in this patient who presented with an increased serum cTnT of 0.461 µg/L (with a peak of >1.1 µg/L on day 7; Table 1
) and whose electrocardiogram was diagnostic for MI on day 3. In contrast, the nonspecific origin of total CK limits its contribution to diagnosis when cardiac and skeletal muscle diseases coexist. We do not know whether the increased serum cTnT was a result of myocardial ischemia, ubiquitous myopathic changes during the progression of the disease, or some combination. Regardless, a cardiac component could clearly be demonstrated.
Changes in fsTnI and/or ssTnI could be influenced by clinical conditions, directly or indirectly, associated with rhabdomyolysis. Acute renal failure, compartmental syndrome, and cardiac abnormalities are common complications that can affect either the release or clearance of sTnI (17). In addition, patients may experience muscle atrophy, regeneration, and/or adaptation over time; all of which could influence the results of serum sTnI analysis. Further work is needed to determine whether changes in fsTnI and/or ssTnI are specific for a given disease (and if so, its severity) and particular muscle types. In addition, the performance of WB-DSA, like any other diagnostic assay using antibodies, is limited by antibody selection and the possibility that modifications of the target protein alter binding affinities and, hence, assay results. It will therefore be necessary to screen different patient cohorts with a variety of antibodies to overcome such limitations. Nevertheless, preferential or selective release of the two isoforms (and their modified products) into blood raises the possibility of improving the differential diagnosis of skeletal muscle injuries or disease, prognosis, and the evaluation of therapeutic effectiveness.
Acknowledgments
This work was supported by grants to S. I. and J. V. E. from the Canadian Institutes of Health Research (MOP 36339 and MT 14375), the Heart and Stroke Foundation of Canada (T-3759), and the Ontario Thoracic Society. We thank Spectral Diagnostics Inc. (Toronto, Canada) for generously providing one of the antibodies for this study.
Footnotes
1 these authors contributed equally to this work; 
References
The following articles in journals at HighWire Press have cited this article:
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  J. A. Simpson, R. Labugger, C. Collier, R. J. Brison, S. Iscoe, and J. E. Van Eyk Fast and Slow Skeletal Troponin I in Serum from Patients with Various Skeletal Muscle Disorders: A Pilot Study Clin. Chem., June 1, 2005; 51(6): 966 - 972. [Abstract] [Full Text] [PDF]
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 B. O'Connell, D. G. Stephenson, R. Blazev, and G. M. M. Stephenson Troponin C isoform composition determines differences in Sr2+-activation characteristics between rat diaphragm fibers Am J Physiol Cell Physiol, July 1, 2004; 287(1): C79 - C87. [Abstract] [Full Text] [PDF]
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 J. A. Simpson, J. Van Eyk, and S. Iscoe Respiratory muscle injury, fatigue and serum skeletal troponin I in rat J. Physiol., February 1, 2004; 554(3): 891 - 903. [Abstract] [Full Text] [PDF]
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