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(Circulation. 1995;92:1927-1932.)
© 1995 American Heart Association, Inc.
Articles |
Serum Cardiac Troponin T and Creatine Kinase–MB Elevations in Murine Autoimmune Myocarditis
From the Department of Pediatrics (K.B., C.P., N.N.), Department of Medical Chemistry and Biochemistry (J.M.), and Department of Pathology at the University of Innsbruck (F.O.), Medical School, Innsbruck, Austria.
Correspondence to Dr Nikolaus Neu, Univ.-Klinik für Kinderheilkunde, Anichstraße 35, A-6020 Innsbruck, Austria.
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Abstract |
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Background We used a murine model of autoimmune myocarditis to investigate systematically whether serum markers of myocardial cell injury, that is, cardiac troponin T (TnT) and the MB isoenzyme of creatine kinase (CK-MB) are useful for the diagnosis of inflammatory heart disease.
Methods and Results Fifty-two A.SW mice were immunized with cardiac myosin to induce myocarditis. The disease was evident on day 12 after the initial immunization in 14 of 22 immunized mice, on day 16 in 7 of 10 mice, on day 19 in 6 of 10 mice, and on day 23 in 5 of 10. The severity of myocarditis increased between days 12 and 16 and remained constant thereafter. TnT was elevated in a considerable number of mice with myocarditis, resulting in a diagnostic sensitivity (number of marker elevations per number of mice with myocarditis) of 0.43 on day 12, 0.71 on day 16, and 0.50 on day 19. CK-MB elevations were not seen on day 12 but resulted in a diagnostic sensitivity of 0.71 on day 16 and of 0.33 on day 19. No elevations of CK-MB or TnT were observed on day 23. All elevations were specific for the disease, as none of the mice lacking myocarditis showed increased markers.
Conclusions In murine autoimmune myocarditis, TnT is a more sensitive marker for the disease than CK-MB. Elevations clearly indicate myocarditis, but negative test results do not exclude the presence of the disease. These data suggest that the determination of CK-MB and, in particular, of TnT, can be useful for the diagnostic evaluation of patients with suspected myocarditis.
Key Words: myocarditis • immunology • coronary disease
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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The determination of serum CK and CK-MB is a well-established and widely accepted method for the laboratory diagnosis and follow-up of myocardial infarction. More recently, assays have been developed allowing the detection of TnT, the tropomyosin-binding protein of the troponin regulatory complex located on the thin myofilament of the contractile apparatus.1 2 Due to the existence of heart-specific TnT isoforms,3 4 these assays have gained increasing diagnostic importance with respect to ischemic myocardial injury, particularly in patients presenting with additional skeletal muscle damage.5 6 7 8 However, it is not clear whether these markers are also useful for the diagnosis of inflammatory heart disease. Although elevations of serum CK and CK-MB have been previously reported in myocarditis,9 10 11 12 13 14 the potential diagnostic value of TnT has not been systematically examined.
In humans, the clinical manifestations and histopathological features of myocarditis vary due to the panoramic pathological mechanisms and genetic predispositions.15 16 17 18 19 Sometimes, the unequivocal diagnosis of myocarditis is not possible, and it is therefore difficult to evaluate diagnostic markers for this disease.20 To circumvent these problems, we used a well-defined murine model of autoimmune myocarditis. In this model, myocarditis is induced by immunization with purified murine cardiac myosin.21 Histopathological signs of inflammation appear between days 10 and 12 after the first immunization and reach a maximal severity 7 to 8 days later.22 The disease is organ specific and cannot be induced with skeletal muscle myosin or nonmuscle myosin.21
In the present study, we compared the serum levels of CK-MB and TnT with the presence and severity of myocarditis at various time points after the immunizations.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Animals
A.SW/SnJ mice were obtained from The Jackson Laboratories and were bred and maintained at the Central Experimental Animal Facility, University of Innsbruck Medical School.
Antigen Preparation and Immunizations
Murine myosin was
prepared from hearts and skeletal muscles
(quadriceps muscle) as previously described.23 Six- to
8-week-old mice were immunized twice at a 7-day interval with 70
µg of cardiac or skeletal muscle myosin in 50 µmol/L sodium
pyrophosphate in a 1:1 emulsion with complete Freund's
adjuvant in both sides of the inguinal region.23
Serum Samples and Histopathology
To correlate serum TnT and
CK elevations with the presence and
severity of myocarditis, blood was exclusively obtained at autopsy.
Briefly, mice were anesthetized with ether and bled
retro-orbitally. Alternatively, blood was obtained by cardiac
puncture. Serum samples were stored individually at -70°C until use.
Immediately after bleeding, mice were killed by cervical dislocation.
Hearts were then removed, fixed in 4% formalin, and paraffin embedded.
Sections were obtained at eight different levels and stained with
hematoxylin and eosin. The diagnosis of myocarditis was established by
the presence of an inflammatory cell infiltrate and myocyte
damage. The disease severity was determined according to a previously
described scoring system21 ranging from 0 to 4 (1
corresponds to infiltration of 
5% of at least one histological cross
section; 2, 5% to 10%; 3, 10% to 20%; and 4, >20%). Differences
in disease severity were analyzed by ANOVA for
multiple-sample comparisons (Bonferroni).
TnT ELISA
Serum TnT levels were determined with the Enzymun
test
(Boehringer Mannheim Corp) according to the manufacturer's
instructions. This sandwich ELISA system is based on two mAbs,
namely, a biotinylated cardiac TnT–specific capture antibody (M7) that
binds to streptavidin-coated plastic tubes and a horseradish
peroxidase–labeled detection antibody (1B10) that cross-reacts
with skeletal muscle TnT. Because the assay has been developed for use
in the human system,24 we had to ensure that it is also
suitable for the murine system. This was achieved by comparing the
reactivity of the mAbs with TnT from mice and humans with the use of
Western blotting. Briefly, human tissue samples were obtained at
autopsy from myocardial septum and intercostal muscles. Murine samples
were obtained from whole hearts and from quadriceps muscles. Crude
extracts were prepared by homogenizing 1 part of minced
tissue in 15 parts (w/v) of Laemmli buffer under reducing
conditions.25 The tissue extracts were then separated in
10% polyacrylamide–sodium dodecyl sulfate
mini-gels (Hoefer Scientific Instruments)26 and
electrophoretically transferred to BA 85 nitrocellulose sheets
(Schleicher and Schuell).27 For
immunostaining,28 the sheets were cut into
strips, blocked with 1% bovine serum albumin in PBS containing
0.1% Tween 20 for 30 minutes, and incubated with either the
biotinylated mAb M7 at concentrations ranging from 15 ng/mL to 600
µg/mL or the horseradish peroxidase–labeled mAb 1B10 at a
concentration of 150 µg/mL. Strips with the mAb M7 were then
incubated for 30 minutes with peroxidase-conjugated streptavidin
(Dako) at a dilution of 1:10 000. Bound antibodies were
detected by conversion of substrate (0.5 g/L, chloronaphthol) in the
presence of 0.01% H2O2. Between each
incubation step, the strips were washed several times with PBS
containing 0.1% Tween 20 and, before reaction with substrate, with PBS
alone.
Because a murine standard for the calibration of the TnT ELISA is not available, it was not possible to determine the absolute concentration of TnT in units of weight per volume. Therefore, the results had to be expressed as the relative TnT increase, which is defined as the ratio between the individual mouse TnT level and the upper normal level. The upper normal level was defined by the mean+SD valuex1.96 of controls. Controls consisted of mice immunized with an irrelevant antigen, ie, skeletal muscle myosin. The ratio yielding absolute diagnostic specificity (no positive test results among mice without myocarditis) at the highest possible sensitivity level, ie, a relative increase of more than 2, was used as the cutoff value for TnT.
Serum CK and CK-MB Assays
Total CK activity was determined at
25°C with the Granutest 15
assay kit (Merck) according to the manufacturer's instructions. The
upper normal value was 131 U/L (mean+SD value of nine untreated
controlsx1.96). Relative CK-MB isoenzyme activity was measured by
fluorescent densitometry after electrophoretic separation of
isoenzymes on agarose gels and incubation with substrate. Separation
and visualization were performed according to the manufacturer's
protocol (REP/EDC system, Helena Laboratories). CK-MB values were
considered elevated if both the relative amount of CK-MB exceeded 4.5%
of the total CK activity and the absolute CK-MB activity exceeded 12
U/L, as these values yielded absolute specificity at the highest
possible sensitivity level.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Adaptation of the TnT ELISA for the Murine System
For the human system, the reliability of the TnT ELISA has been proved.24 Before determining serum TnT levels in mice, the mAbs used with this ELISA had to be tested for their reactivity against murine cardiac and skeletal muscle TnT by Western blotting. As expected, the capture antibody M7 recognized a human cardiac antigen with an apparent molecular weight of 41 kD corresponding to that of TnT (Fig 1B
).24 The minimal antibody
concentration required to detect this antigen was 1.5 µg/mL.
Cross-reactivity with a skeletal muscle antigen of a similar
molecular weight was seen only at a 400-fold higher concentration, ie,
at 600 µg/mL. In the murine system, a similar reactivity pattern was
found but at lower antibody concentrations. The minimal mAb M7
concentration to detect cardiac TnT was 0.15 µg/mL, whereas skeletal
muscle TnT was detectable only at a 2000-fold higher concentration, ie,
300 µg/mL. The horseradish peroxidase–labeled detection antibody
mAb 1B10 equally recognized human and murine cardiac and skeletal
muscle TnT in addition to other antigens (Fig 1C). Therefore,
this mAb
has no isoform specificity. Because the mAbs displayed a similar
reactivity pattern in mice and humans, the ELISA kit is suitable
to detect murine TnT in a heart-specific manner.
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Prevalence of Myocarditis and Histopathology
To analyze
whether TnT and CK-MB are suitable biochemical
markers for inflammatory heart disease, myocarditis was induced by
immunization with cardiac myosin. Sera and heart tissues of the mice
were assayed at various time points. As shown in Table 1,
myocarditis was evident within 12 days after the
first immunization. Because the prevalence of the disease was similar
at all time points tested, it is likely that the onset of the disease
was as early as day 12 in almost all affected mice. Between days 12 and
16, the severity of myocarditis increased significantly and remained
constant thereafter. On day 12, only small, discrete foci of
interstitial inflammation were noticed (Fig 2A and
2B), whereas by day 16, large areas of the
myocardium were infiltrated and damaged (Fig 2C and
2D). As
shown previously,21 skeletal muscle myosin did not induce
myocarditis.
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Serum TnT, CK, and CK-MB Measurements
As shown in Fig
3, individual serum TnT levels were
significantly increased in many but not all animals with myocarditis.
On day 12, 6 of 14 mice with myocarditis had increased TnT levels, and
on day 16, TnT was increased in the majority of diseased animals, ie,
in 5 of 7 mice. Moreover, day 16 showed the highest TnT levels among
all time points studied. On day 19, TnT was elevated in 3 of 6
mice with myocarditis, whereas on day 23, no increased TnT was
detectable regardless of whether the animals were diseased. A few mice
without myocarditis (on days 12 and 16) revealed slightly higher TnT
levels than untreated control animals. Mice immunized with skeletal
muscle myosin showed TnT levels similar to those of untreated controls
(Fig 3).
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Total CK serum activity was elevated in the
majority of animals
immunized with cardiac myosin. The activities ranged from 92 to 1135
U/L (Fig 4) but did not show specificity for myocarditis
(Table 2). The major part of the CK activity was due to
CK-MM (unpublished data, 1994). The specificity was markedly improved
by the determination of CK-MB, particularly if the "percentage
criteria" (CK-MB activity, >12 U/L and >4.5% of total CK
activity) were included. The individual relative CK-MB activities are
shown in Fig 5. Elevations of the CK-MB activity meeting
these percentage criteria were observed in 5 of 7 mice with myocarditis
on day 16 and in 2 of 6 mice on day 19. At all other time points, such
CK-MB elevations were not detectable. TnT and CK-MB elevations occurred
throughout the spectrum of disease severity, ie, 1 to 4 (Figs 3
and 5).
On day 16, a higher disease severity (>2) was always accompanied by a
marker elevation, whereas on days 19 and 23, this was not the case.
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It should be noted that cardiac puncture29 was revealed to be an inappropriate method of bleeding as far as myocardial marker proteins are concerned. This method led to an approximately 10-fold TnT increase and to CK-MB elevations similar to those caused by myocarditis (unpublished data, 1994). These elevations were almost certainly caused by mechanical injury of the heart tissue.
The sensitivities of serum
TnT and CK-MB elevations meeting the
percentage criteria are listed in Table 2. At all time points,
the
elevations were absolutely specific for myocarditis (1.0), as none of
the mice without disease showed increased values. The highest
sensitivity for both markers was observed on day 16. Furthermore, 6 of
the 7 animals with elevated CK-MB also showed elevated TnT. Increased
CK-MB, therefore, was usually accompanied by increased levels of TnT.
Increased levels of TnT, however, did not always correlate with
elevated CK-MB activities.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The present study demonstrates that the determination of serum TnT and CK-MB potentially provides valuable diagnostic information in inflammatory heart disease.
Although the mAbs used with the TnT ELISA had been produced by immunization with the human antigen, they also recognized cardiac TnT from mice. The cardiac-specific capture antibody reacted with the murine antigen at even lower concentrations, perhaps due to autolytic changes of the human tissue. The specificity of mAb M7 for cardiac TnT is consistent with the finding by Katus et al.24 Probably, mAb M7 recognizes an epitope with a largely homologous amino acid sequence in mice and men, which would also be in agreement with earlier reports of amino acid sequence homologies between cardiac TnTs from different species.3 30
Inflammation of the myocardium led to a release of both CK-MB and TnT in a time-dependent manner. Serum elevations of the markers characterize the time course of myocardial cell injury accurately, as the plasma half-lives of TnT and CK-MB are only 2 and 12 hours, respectively.31 32 Therefore, the diagnostic sensitivity of TnT and CK-MB determinations varied during the course of the disease, with the highest sensitivity on day 16 after the initial immunization. In affected mice, the extent of infiltration and myocyte necrosis considerably increases between days 15 and 21.22 After day 23, the inflammatory infiltrate begins to disappear within the next 2 weeks and progressively becomes replaced by scar formation (unpublished data, 1993). It is therefore not surprising that elevations of biochemical marker proteins were found only during a relatively short period of time, ie, between days 12 and 19. The highest levels of TnT and CK-MB were measured on day 16 and thus might reflect maximal myocyte damage. However, even at this time point, the elevations were moderate compared with elevations of these markers seen in patients with acute myocardial infarction.5 8 In our murine model, particularly before day 15, the inflammatory infiltrate is mainly interstitial, and in contrast to the situation in myocardial infarction, myocyte necrosis is not prominent.21 22 This view is also supported by the finding that not all mice with myocarditis had elevated levels of TnT and CK-MB. Furthermore, our study shows that on day 19 and, in particular, on day 23, there is no good correlation between marker levels and disease severity. This situation was expected because marker elevations reflect only acute myocyte damage. However, the severity of myocarditis is equal to the extent of cellular infiltration and not to acute myocyte damage. In the animal model that we used, acute myocyte damage must have occurred between days 12 and 19 because no marker elevations were observed thereafter.
Human myocarditis is frequently caused by enteroviruses and adenoviruses, and the histopathological changes depend on the genetic predisposition of the host, the pathogen, and the time point studied.33 34 In acute myocarditis, the initial histological findings are characterized by polymorphonuclear cells surrounding numerous foci of acute myocyte damage.19 At these time points, the sensitivity of myocardial marker determinations should thus be relatively high. By contrast, in chronic forms where myocyte damage is not prominent and mainly interstitial mononuclear cells are present,19 the sensitivity of marker determinations in general might be lower. In coxsackievirus B3–infected A.SW mice, the histopathological changes are similar to the situation in humans. Substantial myocyte damage occurs a few days after viral infection, whereas later phases of the disease are characterized by diffuse mononuclear cell infiltrate and calcification.35 36 Cardiac myosin–induced myocarditis largely resembles the late phase of the virus-induced disease.21 Recently, the first clinical confirmation of TnT elevations in patients with myocarditis was reported by Franz et al.37
In most animals immunized with cardiac and skeletal muscle myosin, the total CK activity was considerably elevated. The poor specificity of these elevations for myocarditis and the fact that the major part of the CK activity was due to CK-MM suggest that increased CK is mainly a result of local skeletal muscle damage caused by inflammation at the injection sites. Release of CK-MM leads to an additional reduction in CK-MB percentage, thereby further decreasing the sensitivity of this isoenzyme for the detection of myocarditis. In addition, injured skeletal muscle releases not only CK-MM but also CK-MB, particularly if the injury is chronic.38 39 40 A significant elevation in CK-MB percentage also occurred in cardiac myosin–immunized mice without myocarditis between days 19 and 23 (unpublished data, 1994). Reduction of sensitivity and loss of specificity are also seen in clinical routine diagnosis, particularly in patients with skeletal muscle damage and suspected concomitant myocardial injury.41 42 In the present study, elevations in CK-MB activity can, in parallel to the clinical situation, be exploited as indicators for myocarditis only if a defined percentage criterion of the total CK activity is used. These difficulties are not seen when cardiac TnT is used as a marker protein. Although it is conceivable that the persistent skeletal muscle damage at the injection sites led to release of skeletal muscle TnT in parallel to the CK release, such a situation would not have caused false-positive TnT levels, thereby supporting the heart specificity of the TnT ELISA. The slightly higher TnT levels observed in cardiac myosin–immunized mice without myocarditis were most probably due to minimal TnT contaminations of the immunogen gradually released from the injection sites. Although the cardiac myosin preparation was highly purified as checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, it contained trace contaminations of TnT detectable by the Enzymun test (unpublished data, 1994).
In summary, our data suggest that the determination of CK-MB and TnT should be recommended in cases of suspected myocarditis because elevations of these markers definitely support the diagnosis. Due to its higher diagnostic sensitivity, TnT is a more suitable marker for myocarditis than is CK-MB. However, normal TnT or CK-MB values do not exclude the presence of the disease.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by the Austrian FWF Project P 8960. The mAbs and TnT ELISA kit were a kind gift from Dr Hallermayer, Boehringer Mannheim, Penzberg, Germany. The excellent technical assistance of Sabine Joebstl and Martin Lorenz as well as the support of Dr Bernd Puschendorf are gratefully acknowledged. We thank Dr Josef Penninger and Dr Klaus Lewin for critically reviewing the manuscript.
Received February 27, 1995; revision received May 1, 1995; accepted May 3, 1995.
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 A. Braun, B. L. Trigatti, M. J. Post, K. Sato, M. Simons, J. M. Edelberg, R. D. Rosenberg, M. Schrenzel, and M. Krieger Loss of SR-BI Expression Leads to the Early Onset of Occlusive Atherosclerotic Coronary Artery Disease, Spontaneous Myocardial Infarctions, Severe Cardiac Dysfunction, and Premature Death in Apolipoprotein E-Deficient Mice Circ. Res., February 22, 2002; 90(3): 270 - 276. [Abstract] [Full Text] [PDF]
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 R. L. DeBiasi, C. L. Edelstein, B. Sherry, and K. L. Tyler Calpain Inhibition Protects against Virus-Induced Apoptotic Myocardial Injury J. Virol., January 1, 2001; 75(1): 351 - 361. [Abstract] [Full Text]
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 S. C. Smith, J. H. Ladenson, J. W. Mason, and A. S. Jaffe Elevations of Cardiac Troponin I Associated With Myocarditis: Experimental and Clinical Correlates Circulation, January 7, 1997; 95(1): 163 - 168. [Abstract] [Full Text]
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A. Braun, B. L. Trigatti, M. J. Post, K. Sato, M. Simons, J. M. Edelberg, R. D. Rosenberg, M. Schrenzel, and M. Krieger Loss of SR-BI Expression Leads to the Early Onset of Occlusive Atherosclerotic Coronary Artery Disease, Spontaneous Myocardial Infarctions, Severe Cardiac Dysfunction, and Premature Death in Apolipoprotein E-Deficient Mice Circ. Res., February 22, 2002; 90(3): 270 - 276. [Abstract] [Full Text] [PDF]
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