Elevations of Cardiac Troponin I Associated With Myocarditis
Experimental and Clinical Correlates
Background Endomyocardial biopsy is currently the standard method used to diagnose myocarditis. However, it is invasive and has a low diagnostic yield. Because the histological diagnosis of myocarditis requires the presence of myocyte injury, we sought to determine whether measurement of cardiac troponin I (cTnI), which is a serum marker with high sensitivity and specificity for cardiac myocyte injury, could aid in the diagnosis of myocarditis.
Methods and Results To validate this approach, cTnI values were first measured in mice with autoimmune myocarditis. cTnI values were elevated in 24 of 26 mice with myocarditis but were not elevated in any of the control animals (P<.001). Next, cTnI values were measured in the sera from 88 patients referred to the Myocarditis Treatment Trial and were compared with creatine kinase–MB (CK-MB) values measured in the same patients. cTnI values were elevated in 18 (34%) of 53 patients with myocarditis and in only 4 (11%) of 35 patients without myocarditis (P=.01). In contrast, CK-MB values were elevated in only 3 (5.7%) of 53 patients with myocarditis and 0 of 35 patients without myocarditis (P=.27). Thus, elevations of cTnI occurred more frequently than did elevations of CK-MB in patients with biopsy-proven myocarditis (P=.001). Importantly, elevations of cTnI in patients with myocarditis were significantly correlated with ≤1 month duration of heart failure symptoms (P=.02), suggesting that the majority of myocyte necrosis occurs early, and thus the window for diagnosis and treatment may be relatively brief.
Conclusions cTnI was superior to CK-MB for detection of myocyte injury in myocarditis, and cTnI elevations were substantially more common in the first month after the onset of heart failure symptoms.
A simple, highly sensitive and specific test that could accurately detect myocyte injury during the course of myocarditis would be clinically valuable. EMB is the standard method,1 2 3 4 but results of several studies suggest that it may underestimate the true prevalence of disease. Fowles and Mason1 reported that EMB confirmed the diagnosis of myocarditis in only 25% of their patients in whom it was suspected clinically, and observations reported by Parrillo et al5 were similar: criteria for myocarditis were met by EMB in 26% (19/74) of patients with unexplained heart failure. More recently, the MTT4 reported only a 10% incidence of diagnostic biopsies in 214 of 2233 patients with heart failure, no evidence of coronary artery disease, and no other explanation for reduced ventricular function. Either myocarditis is a rare disease or it may be that we have yet to define the cellular markers or pathological findings that provide for accurate diagnosis.6 This may be due to marked interobserver variability in the interpretation of the biopsy findings,4 7 the relatively small area of myocardium sampled by EMB, or the diversity of etiologies that may make the manifestations of myocarditis vary from patient to patient. The timing of EMB is also important, because it is known that the clinical and histological features of myocarditis do not always coincide.8 Thus, it may be that the low incidence of EMB-proven disease reflects our inability to establish the diagnosis of myocarditis by the histological criteria presently in use.
At present, the histological diagnosis of myocarditis requires the presence of a lymphocytic infiltrate and evidence of myocyte injury.6 It is the histological detection of myocyte injury in this setting that often is difficult. In the MTT, this was the criterion for myocyte injury that was the most frequent cause of disagreement among pathologists.4 Thus, the use of other techniques to confirm the presence of cardiac injury, such as antimyosin imaging9 10 or the measurement of serum markers that are specific for cardiac injury, might be useful to augment the sensitivity of EMB in diagnosing myocarditis.
Recently there has been progress in the identification of long-lived serum markers of myocyte injury.11 Elevation of cTnI, which regulates the calcium-mediated interaction between actin and myosin, is a sensitive and highly specific serum marker for cardiac injury.12 Because most of the cTnI is physically associated with the myocyte contractile apparatus,13 it is released slowly into the serum after cardiac injury and can be detected for up to 14 days afterward. This provides a larger diagnostic window for detection of cardiac injury than previously used markers such as CK-MB.
To determine whether elevations in values of cTnI occur during the course of myocarditis, a well-characterized murine model of autoimmune myocarditis14 15 16 was used for experimental validation of the approach. In this model, a T cell–mediated immune response14 is induced that is specific for the cardiac isoform of myosin,17 resulting in autoimmune myocarditis. Because the premise of the MTT was that immunosuppression might improve left ventricular function in myocarditis because immune-mediated myocardial injury contributed to the pathogenesis of left ventricular dysfunction,4 18 this model appeared appropriate to parallel the MTT. The animal studies were performed first because the experimental group was uniform, the kinetics of myocyte injury were well defined, and the entire heart was available for histological analysis. On the basis of our experiments with the murine model, we proceeded to measure cTnI from the sera of patients enrolled in the MTT.
These studies were approved by the Animal Studies Committee at Washington University, St Louis, Mo, and all procedures were performed according to institutional guidelines. Male A/J mice (n=41) were obtained from Jackson Laboratories (Bar Harbor, Me) and were 2 to 4 months of age at the time of experiments. All mice were housed in microisolator cages (Allentown Caging) and fed autoclaved water and irradiated rodent chow ad libitum.
Purification of Mouse Cardiac Myosin
Mouse cardiac myosin was prepared according to a modification of the procedure of Shiverick et al,19 as described in detail previously.14 Briefly, mice were killed by cervical dislocation and their hearts rapidly excised and placed into PBS at 4°C. The tissue was then homogenized, and large connective tissue fragments were removed by centrifugation at 12 000g for 10 minutes. The myosin was precipitated with water, and after removal of actomyosin, the purified myosin was dialyzed into 50 mmol/L sodium pyrophosphate, pH 7.3, at 4°C. Purity of the preparation was assessed by SDS-PAGE under reducing conditions. The myosin concentration was measured by the Bradford method of protein quantitation (Bio-Rad).
Induction of Myocarditis
A total of 100 μg myosin was emulsified in CFA containing Mycobacterium tuberculosis strain H37Ra (Difco), and mice were injected subcutaneously as previously described.14 Control mice received an equal volume of CFA without myosin, which previously has been shown not to induce myocarditis.14 16 Five hundred nanograms of pertussis toxin (List Biological Laboratories) was injected intraperitoneally at the time of the first immunization only into both experimental and control animals. In previous experiments, animals were killed at various times to define the time course of the development of myocarditis (S.C.S., MD, unpublished data, 1990). For these experiments, all mice were killed by cervical dislocation on day 21, and their hearts were rapidly excised and immediately placed into 10% buffered formalin.
After 24 hours of fixation in formalin, the hearts were embedded in paraffin, and serial transverse step sections were made along the entire length of the heart. The sections were stained with hematoxylin and eosin, and the degree of myocarditis was assessed by light microscopy. The histological severity of myocarditis was determined as previously described14 : severe, >50% of myocardium involved; moderate, 10% to 50% of myocardium involved; minimal, <10% of myocardium involved; or normal.
The design of the MTT, methods of patient randomization, patient characteristics, and histopathologic techniques used for the evaluation of myocarditis are described in Mason et al4 and Hahn et al.18 Briefly, patients were eligible to participate in the MTT if they had unexplained heart failure of <2 years' duration with a radionuclide left ventricular ejection fraction of <0.45, EMB-proven myocarditis, and no contraindications to immunosuppression and if they were able to give informed consent for participation in the study. EMB was performed by standard techniques under institutional guidelines, and the specimens were fixed in formalin before being processed, sectioned, and stained with hematoxylin and eosin. The histopathologic diagnosis of myocarditis was made at the patient's referring institution. Each patient's biopsy sample was then reviewed by a panel of seven expert pathologists, and myocarditis was diagnosed according to the Dallas criteria.6 Control patients had unexplained heart failure without evidence of myocarditis on biopsy.
Patient Subgroup Analysis
cTnI and CK-MB values were measured from a subgroup of patients referred for inclusion in the MTT who had remaining serum available for analysis. This group consisted of 53 patients in whom myocarditis was diagnosed according to the Dallas criteria on EMB after review by the MTT pathology panel and in 35 control patients with unexplained heart failure and no evidence of myocarditis on biopsy.
cTnI and CK-MB Measurement
Serum samples were obtained from mice by tail bleeding, according to institutional guidelines. In the MTT patients, serum samples were obtained by venipuncture at the time of EMB. cTnI was measured by an enzyme immunoassay that uses two cTnI-specific MABs with independent epitopes for cTnI. The assay was performed with a preliminary research application on the Stratus analyzer (Dade International, Inc).12 20 The MABs used in this assay have no known species specificity because the amino acid sequence of cTnI is highly conserved. For example, 195 of 210 amino acids are identical between human and mouse cTnI,21 22 and 9 of the 15 amino acid differences represent conservative changes.23 This was confirmed by measuring cTnI in the homogenate of a mouse heart. The amount of cTnI measured per gram of protein was similar to that of human or bovine heart (J.H.L., PhD, unpublished data, 1993). The assay for cTnI has no detectable cross-reactivity with human skeletal muscle troponin I.20 All assays were performed by technicians blinded to the source of the serum. The upper limit of the reference range of the assay for cTnI is <3.1 mg/mL; the lower limit of detection is 1.5 ng/mL. CK-MB (normal, <6.7 ng/mL; lower limit of detection, 2.2 ng/mL) was measured by immunoassay as previously described.12 20
Statistical analysis was performed by Mann-Whitney rank sum test and Wilcoxon signed rank test24 with the use of the StatView (Abacus Concepts) statistical software program on a Macintosh computer. Protein sequence analysis was performed by use of the Nentrez software program and sequence information obtained from GenBank (National Center for Biotechnology Information, Bethesda, Md).
cTnI Is Elevated in Mice With Myocarditis
cTnI values were measured in the sera of mice 21 days after immunization with cardiac myosin, when the histological appearance of myocyte injury was maximal.14 16 cTnI was elevated in all 16 animals with severe myocarditis, in 6 of 7 with moderate myocarditis, and in 2 of 3 mice with minimal myocarditis (Fig 2⇓). Overall, cTnI was elevated in 24 of 26 mice with myocarditis but was not detected in the sera from any of the 15 control mice immunized with CFA alone. Thus, cTnI values were elevated only when histological evidence of myocarditis was present. This difference between the cTnI values in mice with myocarditis and control mice was statistically significant (P<.0001). The time course of cTnI elevation follows the kinetics of histologically detected myocyte injury, as cTnI can be detected as early as day 12 after immunization (Fig 1⇑, and S.C.S., MD, and P.M. Allen, PhD, unpublished data, 1990). Although there was a trend for cTnI elevations to parallel the histological severity of myocarditis, the differences in cTnI values among the animals with minimal, moderate, and severe myocarditis did not achieve statistical significance.
cTnI Elevations in Patients With Myocarditis
Values of cTnI and CK-MB were measured in the sera of patients with a histological diagnosis of myocarditis and compared with values measured in patients with unexplained congestive heart failure but no evidence of myocarditis on EMB (Fig 3⇓; Table).⇓ cTnI values were significantly greater in patients with myocarditis than in control patients (P=.01). In contrast, CK-MB values were not significantly greater in patients with myocarditis than in control patients (P=.27). Among the 53 patients with myocarditis, 18 (34%) were found to have elevated cTnI values, but only 3 (5.7%) had elevated CK-MB values. Thus, the frequency of a positive cTnI level was significantly greater than a positive CK-MB value in patients with myocarditis (P=.001). In the control patients, differences in the frequency of positive cTnI and CK-MB values were not significantly different, as only 4 (11%) of 35 patients were found to have an elevated cTnI level, and none of these 35 patients had elevated CK-MB values (P=.07). When these data are used, the sensitivity of an elevated cTnI for the diagnosis of myocarditis is 0.34, the specificity is 0.89, and the positive predictive value is 0.82.
Relationship of the Duration of Heart Failure Symptoms to Elevation of cTnI
To determine the relationship between the duration of heart failure symptoms and the extent of cTnI elevations, we analyzed cTnI values in 32 of the 53 patients who had information available on the duration of their heart failure symptoms. cTnI values were significantly higher in the 20 patients with symptoms lasting ≤1 month (Fig 4⇓) than in the 12 patients whose symptom duration was >1 month (P=.02). Eleven of the 20 patients with symptoms for <1 month had values of cTnI ≥3.1 ng/mL, whereas only 1 of the 12 patients with a longer duration of symptoms had an elevated level of cTnI.
Relationship of the Histopathologic Pattern and Severity of Myocarditis to Elevation of cTnI
There was no apparent quantitative relationship between the extent of the elevations observed in cTnI and the severity of myocarditis detected pathologically in mice. To determine whether there was a relationship between the pattern of myocarditis and the extent of cTnI elevations in patients, we correlated cTnI values with the histological severity of myocarditis. Of the 18 patients with elevated cTnI values, 6 had focal, 8 had diffuse, and 4 had borderline myocarditis (Fig 5⇓). The mean cTnI level was increased in the group of patients with diffuse myocarditis (25.5 ng/mL) compared with the cTnI values in the patients with focal (8.9 ng/mL) or borderline myocarditis (5.1 ng/mL), but this difference did not achieve statistical significance.
Our experimental data suggest that detection of myocyte injury during the course of myocarditis may be facilitated by the measurement of cTnI. This approach is feasible and most likely to be of benefit early after the onset of symptoms. cTnI values were significantly higher in patients with myocarditis than in the control patients without myocarditis (P=.01), whereas there was no significant difference in CK-MB values (P=.27). Importantly, elevated cTnI values were more frequent in patients with myocarditis than were elevated CK-MB values (P=.001; Table). Thus, measurement of cTnI was significantly better for identifying patients with histological evidence of myocarditis. Such a result is in keeping with data that show that cTnI has sensitivity comparable to CK-MB for the detection of cardiac injury.13 In addition, cTnI manifests a slow release over time, which broadens the window during which elevations can be detected,20 and has unique specificity for cardiac injury.12 Our study results are also consistent with the results of previous studies26 27 28 29 with CK-MB that have documented its lack of reliability for the detection of myocarditis, perhaps because of its relatively brief persistence in serum after myocardial injury. These data suggest that in patients with unexplained heart failure, an elevated cTnI level suggests ongoing myocyte injury and the possibility of myocarditis. Other causes of myocyte injury, such as ischemia, toxins, and infiltrative diseases, could raise cTnI values, because detection of cTnI is not specific for inflammatory-mediated myocyte injury.
Timing clearly emerges from the clinical data as one of the critical parameters for the efficacy of cTnI measurement to detect myocarditis. Analysis of the relationship between the duration of heart failure symptoms and the extent of cTnI elevation revealed that cTnI was most likely to be elevated in patients with myocarditis early (within 1 month) after the onset of symptoms. This finding is consistent with the hypothesis that the majority of myocyte injury and necrosis may occur early in the patient's course, reflecting either the kinetics of the injuring stimulus or possibly the inflammatory response to myocyte injury and initiation of the process of tissue repair. If this is the case, both diagnostic procedures that detect acute myocyte injury and therapies aimed at reducing myocyte necrosis would likely be most effective early during the course of the disease. Detection of lymphocytes and injured myocytes on EMB later on might reflect previous rather than ongoing injury. We cannot exclude the possibility that detection would have been improved had serial measurements of cTnI been performed.
Many of our patients with myocarditis did not have elevated cTnI values. The inability of cTnI measurement to detect more patients with myocarditis is most likely multifactorial. Although viral infection is the probable cause in most cases of myocarditis, several different viruses have been implicated,4 30 31 32 each with unique kinetics of pathogenesis. In addition, an autoimmune-mediated response to endogenous cardiac proteins, which is likely to have a different time course than viral myocarditis or experimental murine autoimmune myocarditis, is another possible cause in some cases of human myocarditis. Importantly, in contrast to our experimental studies, our patients presented up to 2 years after the onset of clinical symptoms. By then, the majority of necrosis may have already occurred. Thus, given the long duration of disease, the late evaluation, and individual variability in the release and clearance of cTnI in response to the kinetics of injury, it is not surprising that a solitary measurement of cTnI did not detect elevations in all of the patients with myocarditis. Finally, we cannot be sure, despite the presence of inflammatory cells and necrotic myocytes, that ongoing damage was present or was severe enough to cause elevation of cTnI.
This contrasts with our findings that 24 of 26 mice with myocarditis had elevated cTnI values. In the murine model, the animals were genetically identical and had uniform immune responses to an identical and controlled stimulus for myocyte injury, thus making the probability of detecting cTnI greater. In addition, the time course of injury was defined and consistent, and the entire heart was available for histological analysis. These animal data are similar to those reported by Bachmaier et al,33 in which measurement of cardiac troponin T was used to detect cardiac myocyte injury in murine autoimmune myocarditis. Clearly, the detection of myocarditis in humans is significantly more complex than in defined and controlled animal models.
These are the first clinical data thus far reported in which cTnI was used to evaluate patients with myocarditis. Solitary measurements of a serum marker with high sensitivity and specificity for cardiac myocyte injury, such as cTnI, at the time of evaluation may be an important first step toward finding new approaches to the diagnosis of myocarditis but may not be adequate in and of itself. Additional data will be necessary to assess whether more frequent and/or earlier samples for measurement of cTnI would improve sensitivity or whether other approaches such as the use of anti-myosin antibodies,9 10 which would detect both old and ongoing necrosis, might be superior. The sensitivity of anti-myosin scintigraphy for detection of myocarditis in other series is better than what we have reported for cTnI (0.83 versus 0.34), but cTnI elevations in the present study appear to be more specific (0.89 versus 0.53). Thus, cTnI measurement may be useful to evaluate the possibility that a patient has ongoing myocyte necrosis. The lack of elevations of cTnI would favor the likelihood that acute myocyte necrosis, which may have occurred in the past, was no longer present. cTnI elevations were detected in only 4 of 35 control patients who presented with unexplained heart failure and no evidence of myocarditis on EMB. It is possible that these 4 patients had another form of myocyte injury not attributable to myocarditis, because cTnI elevations are not specific for myocarditis. It is also possible that the histological evidence of myocarditis was missed on EMB because of sampling error. It is unlikely that these cTnI elevations represent damage to tissue other than myocardium because thus far, false-positive elevations of cTnI have not been reported even in 215 patients studied with chronic renal failure or acute skeletal muscle injury.12 Furthermore, the origin of cTnI suggests that elevations should be highly specific for cardiac injury.11
It is interesting that cTnI elevations did not correlate with the histological severity of myocarditis either in patients from the MTT or in mice with autoimmune myocarditis. Several explanations for this finding are possible. The kinetics of myocyte injury are prolonged, making it difficult to select the ideal conjoint sampling interval for both EMB and cTnI measurement when only one sampling time of each is available. Variations in cTnI release related to the stage of disease would add to this difficulty. Finally, the histological pattern of myocarditis may not always be a marker of the severity or extent of myocyte injury. Although lymphocytic infiltration may be intense, it is the extent of necrosis that would be related to elevations in cTnI in the systemic circulation, and it is unclear that there is a direct relationship between the number of infiltrating lymphocytes and the extent of myocyte injury on EMB.
At the present time, EMB remains the standard test for the diagnosis of myocarditis.4 It is limited by the small amounts of tissue that can be obtained, which is particularly important in detecting cardiac myocyte necrosis. It is likely that sensitive tests specific for myocyte injury, such as cTnI, would aid in the diagnosis of myocarditis.
Selected Abbreviations and Acronyms
|CFA||=||complete Freund's adjuvant|
|cTnI||=||cardiac troponin I|
|MTT||=||Myocarditis Treatment Trial|
Dr Smith was a Howard Hughes Physician Postdoctoral Fellow. This work was supported by grants from the National Institutes of Health and the Council for Tobacco Research. We thank Vonnie Landt and Sharon Porter for their invaluable assistance with the cTnI and CK-MB assays.
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994, and published in abstract form (Circulation. 1994;90[pt 2]:I-547).
- Received May 2, 1996.
- Revision received August 14, 1996.
- Accepted August 24, 1996.
- Copyright © 1997 by American Heart Association
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