Cardiac specific troponin-I release in canine experimental myocardial infarction: Development of a sensitive enzyme-linked immunoassay

J Mol CeU Cardio119, 999-1010 (1987)

Cardiac Specific Troponin-I Release in Canine Experimental Myocardial Infarction: Development of a Sensitive Enzyme-Linked Immunoassay Bernadette Cummins

and Peter Cummins

British Heart Foundation Molecular Cardiology Unit, Clinical Research Block, University of Birmingham, Birmingham, UK (Received 9 March 1987, acceptedin revisedform 13 August 1987) B. CUMMINSANDP. CUMMINS.Cardiac Specific Troponin-I Release in Canine Experimental Myocardial Infarction: Development of a Sensitive Enzyme-Linked Immunoassay. Journal of Molecular and Cellular Cardiology (1987), 19, 999-1010. A canine model of experimental myocardial infarction has been used to investigate the release of troponin-I as a specific diagnostic indicator of cardiac necrosis. An enzyme-linkedimmunoassaywas established to detect canine cardiac troponin-I in serum. Polyclonal antisera to cardiac troponin-I showed low cross-reactivity with skeletal muscle troponin-I which was completely removed by immunoadsorption. The cardiac specific ELISA time was 5 to 6 h. Assaysensitivitywas 4 ng cardiac troponin-I/ml with an upper limit of 200 ng/ml in neat serum. Mean normal circulating levels of cardiac troponin-I were 15.6 ng/ml compared with an estimated 11 ng/ml in man [Cummins, B. et al. Am HeartJ 113, 1333-1344 (1987)]. After experimental infarction, cardiac troponin-I was detectable within 4 to 6 h and peaked between 10 to 16 h post-ligation. Cardiac specific creatine kinase-MB isoenzyme was released with a similar initial time course. Mean peak cardiac troponin-I and CK MB were elevated 6- and 10-fold respectively. Cardiac troponin-I levels were elevated for upto 200 h post-ligation compared to a maximum of 100 h for CKMB. The prolonged time course of troponin-I release was comparable to that seen clinically [Cummins, B., et al. Am Heart J 113, 1333-1344 (1987)]. Histochemicalinfarct size correlated well with CK--MBbut less so with troponin-I release. This may reflect the complexnature ofintracellular troponin-I degradation and lossfrom necrotic cardiac tissue. KEY WORDS: Troponin-I ; Infarction ; Enzyme-linkedimmunoassay.

Introduction

A n u m b e r of methods are currently employed in the diagnosis a n d assessment of extent of m y o c a r d i a l cell damage. Clinically these include electrocardiographic, radioisotopic a n d chemical techniques in a d d i t i o n to consideration of p a t i e n t history. A serious limitation with m a n y of these tests is the relative lack of cardiac specificity. This has been increasingly highlighted in the case of chemical assays such as that for creatine kinase MB isoenzyme ( C K - M B ) . A l t h o u g h C K - M B is located p r e d o m i n a n t l y in the heart a n d is elevated post myocardial infarction, it is k n o w n that elevations due to the presence of C K - M B in skeletal tissues are seen after severe skeletal muscle t r a u m a [2, 13]. I n consequence atten-

tion has focused on more cardiac specific markers as diagnostic agents. T h e contractile proteins of the myofibril exist in a variety of isotype forms some of which are t h o u g h t to be cardiac specific [-for review see 32]. T r o p o n i n - I ( T n - I ) , the inhibitory protein of the t r o p o n i n regulatory complex is k n o w n to exist in three different isotype forms representative of cardiac, fast a n d slow skeletal muscle [31, 36]. I n the r a b b i t there is considerable p r i m a r y sequence homology ( a b o u t 40%) between the three forms with the cardiac isotype (molecular weight 22500 daltons) possessing a n additional 26 residue sequence on the N - t e r m i n u s [10, 11, 35, 36]. Evidence suggests that the same general relationship between the cardiac a n d skeletal isotypes applies to all m a m m a l i a n

Please address all correspondence to: P. Cummins, Molecular Cardiology Unit, Department of Cardiovascular Medicine, Clinical Research Block, Universityof Birmingham,Birmingham,B15 2TH, UK 0022-2828/87/010999 + 12 $03.00/0

9 1987 Academic Press Limited

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species studied to date [3, 8, 12]. The cardiac isotype has been shown to be uniquely located in the heart in a variety of mammalian species including man, and is moreover the only T n - I isotype present in the heart [4, 8, 12]. As a consequence of its heart specificity, detection of circulating cardiac T n - I has been employed in a recent clinical study to diagnose myocardial infarction in an unambiguous manner [4]. A radioimmunoassay to determine serum Tn-I was employed in the latter investigation capable of measuring near to normal circulating levels. A prominent feature to emerge from the clinical studies was the prolonged release profile of T n - I with circulating levels elevated above normal for several days post-infarction possibly reflecting release from two intracellular pools [4]. In order to investigate in more detail the potential for using cardiac T n - I as a diagnostic agent both for in vitro blood immunoassay and for possible in vivo use via the injection of radiolabelled antibodies as has been employed for myosin [16, 17] it was decided to establish a canine model for experimental studies. In the following investigation we report the establishment of a sensitive and specific enzyme-linked immunoassay (ELISA) for cardiac Tn-I and its application in a canine model of myocardial infarction. Some aspects of these results have been presented briefly in abstract form previously [5,

6]. Materials and Methods

Tissue sources Canine ventricular and skeletal tissues used for protein preparations were excised immediately after death, frozen in liquid nitrogen and stored at --25~ until use.

Preparation of Tn-I Cardiac and skeletal T n - I were prepared using the affinity chromatographic procedure described previously [3/] with some modifications [4]. Tn-I preparations at concentrations of 0.5 to 1 mg/ml were only sparingly soluble at neutral pH and required high ionic strength buffers for complete solubilisation. Consequently after elution from troponin-C affinity columns, all T n - I preparations were

dialysed directly against 0.5 M sodium chloride, 20 mM tris-HC1, 60 mM 2-mercaptoethanol pH 7.5 to remove traces of urea, and stored in this buffer at --25~ In some cases, insolubility of stored preparations was associated with the appearance on SDS gels of higher molecular weight, mainly dimer, species. This insolubility could be prevented and the dimers lost on addition of fresh 2mercaptoethanol to stored solutions and presumably reflected dissociation of intermolecular disulphide bonds. At concentrations of less than 0.1 mg/ml, T n - I was completely soluble in solutions of physiological ionic strength at neutral pH.

Polyacrylamide gel electrophoresis This was carried out essentially as described by Weber and Osborn [34]. Samples were electrophoresed in the presence of0.1% (w/v) sodium dodecyl sulphate (SDS), 0.1 M sodium phosphate buffer p H 7.0 in 10% (w/v) acrylamide, 0.27% (w/v) bisacrylamide gels. 60 mM 2-mercaptoethanol was included in all T n - I samples to reduce dimer formation which was prevalent at the usual get sample concentrations (15 raM) employed.

Polyclonal antisera production This was carried out as described previously [4]. Essentially, increasing doses of Tn-I were administered intravenously into rabbits after priming intramuscular injections of Tn-I in both complete and incomplete Freund's Adjuvant. Titres of antibody were determined 3 days after the final intravenous injection and, if satisfactory, larger bleeds were taken. Immunoadsorption of anti-cardiac T n - I sera on mixed canine fast and slow skeletal Tn-I affinity columns was carried out at 4~ using standard procedures. Skeletal troponinI was bound to Sepharose-4B using cyanogen bromide at a level of 5 mg Tn-I/g Sepharose. 18 to 20 ml of a 50% ammonium sulphate fraction of neat antisera was applied to a 5 g (wet weight) column of skeletal T n - I Sepharose in 0.14 g sodium chloride, 0.01 M potassium phosphate buffer, pH 7.1 (physiologically buffered saline; PBS). The void fraction was used in the ELISA. Bound skeletal cross-reacting antibodies were removed by elution with 3 M potassium thio-

Troponin-I in Myocardial Infarction

cyanate, 10 m i sodium phosphate buffer, p H 7.1 and discarded.

Enzyme-linked immunosorbent assay (ELISA ) The E L I S A is a competitive assay in which the level of Tn-I is determined by its ability to inhibit the binding of a fixed amount of antibody to T n - I which has been adsorbed onto plastic microtitre plates. The optimum antiserum dilutions and plate-coating concentrations for bound antigen were determined by "checkerboard" titrations over a range of different antiserum and antigen concentrations. Polyvinyl microtitration plates (Falcon, Becton Dickinson Ltd, UK) were coated overnight at 4~ with T n - I (50 #1 per well of a 0.5 #g/ml solution) in 0.05 i sodium carbonate buffer pH 9.6. Protein binding sites remaining after Tn-I incubation were blocked using 2% (w/v) bovine serum albumin (BSA) in coating buffer. Plates could be stored at this stage for upto 1 month at 4~ Prior to use plates were washed with PBS, 0.5% (v/v) tween-20 and blotted dry. T n - I standards were diluted in PBS, 0.5% (v/v) tween-20, 0.5 M potassium chloride, 1% (w/v) BSA down to concentrations of 0.1 mg/ ml and at lower concentrations in the same buffer without potassium chloride. Primary and secondary antibodies were diluted in PBS, 0.5% (v/v) tween-20, 1% (w/v) BSA. All assay procedures were carried out at room temperature. Pre-incubation of equal volumes of T n - I standards (0.5 to 100000 ng/ml) or unknown samples and cardiac Tn-I antiserum was carried out in plastic tubes (10 x 65 mm) for 2 to 3 h. The incubation mixture was then applied to cardiac T n - I coated plates (50 #l/well in triplicate) and left for 1 h. Unbound antibody and antigen/ antibody complex were removed by washing the plate with PBS, 0.5% tween-20 before incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Tissue Culture Services, UK) at a dilution of 1:10000 (50/zl/well) for 1 h. After washing the plate, solid phase bound H R P activity was determined with 0.015 i ophenylene diamine, 0.008 i hydrogen peroxide in 100 mM citrate phosphate buffer pH5 (50 #l/well). After 20 rains, 6 N sulphuric acid (50 #l/well) was added and the absorbance at 492 nm determined in a manual plate

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reader (Titertek Uniskan, Flow Laboratories, UK). Blank values were obtained by substituting the buffer for Tn-I standards and nonimmune serum for antibody. These were subtracted from all Tn-I standard values when plotting the calibration curve. Non-specific binding of test serum samples was determined by replacement of antiserum with nonimmune serum. Maximum b'nding of antibody in the assay was determined in the absence of Tn-I.

Experimental myocardial infarction Adult male or female dogs (13 to 33 kg) were premedicated with acetylpromazine (0.2 rag/ kg intramuscular), and 600 #g of atropine sulphate (intramuscular) 30 mins prior to anaesthetising with pentobarbital sodium (30 mg/kg intravenously). Intubation with a 9 to 10 m m cuffed endotrachael tube was performed to allow positive pressure ventilation of the lungs and 500 to 1000 ml Ringerlactate solution, depending on the size of the animal, infused throughout the operation as fluid replacement therapy. A left lateral thoracotomy was made through the 5th intercostal space to reveal the heart. The pericardium was dissected below the phrenic nerve to expose the major left ventricular coronary arteries, and a pericardial cradle constructed to allow temporary promotion and support of the heart. A ligature was placed around the left anterior descending coronary artery, usually just below the first major branch. After allowing 30 mins for stabilization of general haemodynamic function, 4 ml of 1% lignocaine hydrochloride was given immediately before ligation. Further doses oflignocaine hydrochloride and pentazocine were given as required during the first 24 h of close supervision.

Blood collection and storage An indwelling jugular catheter, exteriorised at the back of the neck, was used for the removal of blood samples from all animals. Samples were removed every 4 h for the first 24 h and at increasing intervals thereafter for upto 10 days post-ligation. These were centrifuged immediately after collection and the serum stored at - 2 5 ~ or where possible in liquid

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nitrogen: All serum samples were fracti0nated and assayed for C K - M B activity within 5 days of collection. Tn-I could be assayed for upto 6 months after collection without any decline in levels.

Infarct visualisation At the end of the experimental period animals were terminated with an overdose of pentobarbital sodium. The heart was rapidly removed and washed in cold saline before being partially frozen at --25~ Saline was used to fill both ventricular chambers of the heart to retain the overall shape whilst freezing. The partially frozen heart was sliced transversely into 3-5 mm thick sections and incubated in 0.5 mg/ml nitroblue tetrazolium, 0.1 M Sorensen's phosphate buffer pH 7.4 [25] for 20 mins at 37~ Normal myocardium stained dark blue whilst necrotic tissue remained unstained. A tracing was then made on transparent film of the outline of the myocardial chambers and the area of necrosis in each slice. This was transferred to graph paper, cut out and weighed and the area determined by reference to a weight/area calibration curve. The mean area value of the dorsal and ventral aspects of each slice together with the measured slice thickness was used to calculate the volume of whole heart, left ventricle and infarct zone. The values obtained by this method were compared with those determined by weighing the normal and infarcted tissue after dissection of each slice. In both methods the septum was divided on the basis of proportional right and left ventricle free wall thickness. Infarct sizes determined by both methods gave comparable results.

Creatine kinase M B isoenzyme assay Canine serum C K - M B isoenzyme was separated from C K - M M and BB using ionexchange chromatography. 0.25 ml serum was applied to a column (1.4 cm x 0.7 cm diameter) of DEAE-Sepharose DCL-6B-100 pre-equilibrated with 50 mM tris-HC1, 30 mM sodium chloride pH 7.5. The M M fraction was eluted with 4 x 5 ml of equilibrating buffer. The MB fraction was eluted with 4 ml of 50 mM tris-HC1, 120 mM sodium chloride pH 7.5, and the BB fraction with 2 ml of

50 mM tris-HC1, 300 mM sodium chloride pH 7.5. The MB fraction was assayed for CK activity at 30~ using an N-acetylcysteine (NAC) activated system (Boehringer Corporation). Precipath E control serum (Boehringer Corporation) was used for standardisation. Evaluation of fraction purity was established using agarose gel electrophoresis.

Agarose gel electrophoresis Fractionated CK isoenzymes were concentrated (approximately 50-fold) using miniconcentrators (Centricon TM 30 microconcentrators, Amicon Corporation, UK). Whole canine serum and concentrated serum CK isoenzyme fractions were electrophoresed on glass plates (8.4 x 9.3 cm) in 1.2% (w/v) agarose in 25 mM veronal buffer, 5 mM EDTA, 3.8 mM sodium azide pH 8.6 essentially according to the method of Ogunro et al. [24]. 20/A samples were allowed to diffuse into the agarose using a plastic film overlay with sample slots and electrophoresed in an LKB Multiphor flat bed electrophoresis system for 1 h at a constant current of 30 mA per plate. After electrophoresis the gel was overlayed with 10 ml of 0.6% agarose containing 50 mM tris, 28 mM magnesium acetate, 18 mM glucose, 20 mM phosphocreatine, 20 mM AMP, 2.6 mM ADP, 80 I U hexokinase, 40 IU glucose-6phosphatedehydrogenase [1] and incubated at 37~ for 1 to 1.5 h. Creatine kinase isoenzymes in the gel were visualized and photographed under ultraviolet light.

Results

Tn-I preparation Canine Tn-I was prepared from ventricular and skeletal muscles as outlined in the Materials and Methods. The yield of Tn-I from cardiac muscle was 0 . 6 3 _ 0.26 mg/g similar to that for mixed skeletal muscle at 0.42 ___0.1 mg/g. When characterised by SDS gel electrophoresis, cardiac Tn-I preparations migrated as a single band (Fig. 1). Skeletal Tn-I preparations also migrated as a single component (Fig. 1) although slightly faster than the cardiac form in keeping with the smaller molecular wieght of 19 800 compared to 22 500 daltons [36]. No signs of proteolytic

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degradation as would be evidenced by smaller peptides were apparent in preparations from fresh cardiac or skeletal muscle.

Cardiac specificity of antisera Antisera to cardiac troponin-I were raised by injection of the cardiac isotype which displays considerable primary sequence homology with the fast and slow skeletal muscle isotypes [36]. The degree of antisera cross-reactivity with the skeletal isotypes was determined using mixed fast and slow skeletal T n - I as the competing free antigen in the ELISA (Fig. 2). Cross-reactivity of antisera was variable but generally low, being about 10% reactive at 250 ng skeletal T n - I / m l and 25% reactive at 100 000 ng Tn-I/ml. This upper level was well beyond the useful working range of the cardiac assay (see below). However, in order to achieve strict cardiac specificity and to avoid subsequent problems arising from possible substantial release of skeletal Tn-I after thoracic surgery, the cross reacting antibodies were removed by immunoaffinity adsorption. Anti cardiac Tn-I serum was adsorbed by passage through a mixed fast and slow skeletal muscle T n - I immunoaffinity column. Comparison of calibration curves using both cardiac and skeletal Tn-I as competing antigen and carried out with pre- and postimmunoadsorbed antiserum (Fig. 2) showed that no detectable cross-reactivity of the postadsorbed antiserum with skeletal T n - I was present upto levels of 100 000 ng/ml.

I00 1,000 I0000 I 0 0 0 0 0 Tn-I (ng/ml.)

F I G U R E 2. ELISA response curves of anti-canine cardiac Tn*I serum with canine cardiac and skeletal Tn-I. Assays were conducted as described in the Materials and Methods both before and after antiserum was absorbed on a canine skeletal Tn-I immunoaffinity column. A Pre-absorption serum at dilution of 1 : 80 000 reacted with cardiac Tn-I; 9 Post-absorption serum at dilution of 1 : 50 000 reacted with cardiac Tn-I; O Pre~ absorption serum at dilution of 1 : 8 0 0 0 0 reacted with skeletal T n - I ; 9 Post-absorption serum at dilution of 1 : 50 000 reacted with skeletal Tn-I.

ELISA for cardiac Tn-I A final post-adsorbed antiserum dilution of 1 : 50 000 and plate coating concentration of 0.5 #g/ml gave optimal assay sensitivity. (For comparison, comparable assay sensitivity, but not cardiac specificity, was obtained with a dilution of 1 : 8 0 0 0 0 in the case of preadsorbed serum). A calibration curve was established using cardiac T n - I concentrations ranging from 0.5 to 10000 ng/ml. Using skeletal T n - I adsorbed rabbit anti-canine serum a calibration curve was constructed from 10 randomly selected assays conducted over a 9 month period (Fig. 3). The detection limit (the lowest concentration of cardiac Tn-I required to inhibit antibody binding at a level of two standard deviations different from maximum binding) was 4 ng cardiac T n - I / m l with a working range upto approximately 200 ng/ml. Levels in excess of 200 ng/ml could be determined by dilution to fall within the working range. Minor variation between

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Tn-I (ng/mt) FIGURE 3. ELISA calibration response curve for canine cardiac Tn-I. Assayswere conducted as described in Materials and Methods with skeletal Tn-I adsorbed rabbit anti-canine cardiac Tn-I serum at a dilution of 1 : 50000. Results of 10 assays are shown. Dashed lines indicate between assay variation ( + 1 S.D.). plates was controlled by inclusion of m a x i m u m b i n d i n g a n d non-specific b i n d i n g references on all plates for each assay. T h e assay procedure took 5 to 6 h to complete. Attempts were m a d e to reduce this time by shortening the p r e - i n c u b a t i o n period b u t this resulted in increased assay variability a n d decreased sensitivity. Increasing the prei n c u b a t i o n t e m p e r a t u r e to 37~ in a further a t t e m p t to reduce assay time also produced the same effects.

CK-MB isoenzyme T h e relative levels of C K - M B isoenzyme in the canine m y o c a r d i u m (less t h a n 2% of total CK) are m u c h lower than in m a n (see Discussion). I n order to q u a n t i t a t e the serum levels of C K - M B released post-infarction in the canine model it was therefore decided to preparatively isolate C K - M B from serum prior to enzyme assay. Q u a n t i t a t i o n of such relatively low a m o u n t s by densitometry after agarose gel electrophoretic separation would be liable to considerable error. Attempts to separate canine C K - M B using commercially

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FIGURE 4. Agarose gel electrophoresis of canine CK isoenzymes.Agarose gel electrophoresis was conducted as described in Materials and Methods. (a), (c). (g), (j), (k) : control sample ofMM, MB and BB isoenzymes; (b), (e): canine serum post-infarction; (d) : normal canine serum ; (f), (h): canine cardiac muscle whole tissue extract; (i): canine skeletal muscle whole tissue extract; (1): fractionated CKMM isoenzyme; (m) : fractionated CK-MB isoenzyme; (n) : fractionated C K - B B isoenzyme; Macro-CK is immunoglohulin-CKcomplex [37]. available ion-exchange c h r o m a t o g r a p h y kits were unsuccessful. T h e major problem was the presence of C K - B B isoenzyme which was present in both n o r m a l a n d post-infarction serum [Fig. 4(b), (d), (e)] a n d which invariably c o n t a m i n a t e d the MB fraction after c h r o m a t o g r a p h y as revealed by agarose gel electrophoresis. T h e precise source of this C K - B B was not clear as e x a m i n a t i o n of crude extracts of canine cardiac and skeletal muscle revealed detectable levels of C K - B B in both [Fig. 4(f), (h), (i)]. C K - M B was however confined to cardiac muscle. Most clinical diagnostic kits are designed to resolve C K - M B from C K - M M / M B mixtures (assuming negligible presence of C K - B B ) . However, c o n t a m i n a t i o n of C K - M B fractions with C K - B B occurred even in those kits examined which were designed to resolve all three isoenzymes, This m a y have resulted from slight differences in charge of canine

Troponin-I in Myocardial Infarction

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F I G U R E 5. C a r d i a c T n - I serum levels post-infarction. Release profiles of i n d i v i d u a l animals are indicated with coronary a r t e r y ligation at zero time. Dotted line indicates u p p e r limit of n o r m a l levels.

when compared with human isoenzymes although the same problems were encountered with a standard mixture of rabbit CK

isoenzymes. Consequently, the canine MM, MB and BB isoenzymes were separated using elution conditions established by experiment

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with non-commercial columns. Under the conditions outlined in the Materials and Methods, all three major isoenzyme fractions were resolved [-Fig. 4(1), (m), (h)] and the activity of the MB fractions measured kinetically.

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Normal serum Serum from seven control animals not subjected to experimentation was assayed for cardiac Tn-I and C K - M B isoenzyme levels. Mean cardiac Tn-I was 15.6 + 4.5 ng/ml (range 9.3-21.5 ng/ml). Pre-surgery blood levels in animals used for the infarction study were not significantly different with a mean of 12.9 + 4.7 ng/ml (range 5.0-20.5 ng/ml) while the sham animals had levels within the normal range with a mean of 15.3 + 3.9 ng/ ml (range 11.1-19.0 ng/ml). Serum C K - M B levels in non experimental animals were low with a mean of 4.7 + 2.0 IU/1 (range 2.3-8.9 IU/1). In animals used for infarction studies pre-surgery levels were 7.0-t- 6.5 IU/I (range 0.114.9 IU/1) while in the sham animals C K - M B levels were 10.0-t- 5.1 IU/1 (range 6.815.9 IU/1). The slightly higher C K - M B levels in experimental animals may have been due in some unknown manner to samples being taken after induction of anaesthesia. It was noted that mean post-operative levels of 6.3 IU/1, within the normal range, were present in the sham animals.

Acute myocardial infarction O f the 12 animals used in the study, three died post-ligation due to ventricular fibrillation. Three animals acted as shams with full operative procedures including all preliminary intramuscular injections being carried out but no ligation taking place. A rise in Tn-I levels above control values was detectable between 4 and 6 h post~ ligation (Figs 5 and 7). Levels continued to rise attaining peak values between 10 to 16 h after ligation. The mean peak level for all animals of 100.7 + 61.2 ng Tn-I/ml (range 44-217 ng/ml) was more than six times greater than the mean control level and occurred at 12 h post ligation (Fig. 7). In most animals Tn-I levels had returned to preoperational or near pre-operational levels by about 200 h post-ligation (Fig. 5).

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FIGURE 7. Mean cardiac Tn-I and CK-MB isoenzyme levels post-infarction. Release profiles are mean for all animals with coronaryartery ligation at zero time. 9 cardiac Tn-I ; 9 CKMB isoenzyme; (a) mean release profiles over 235 h; (b) mean release profiles over first 50 h. C K - M B isoenzyme levels rose above preoperation levels between 3 and 6 h postligation (Fig. 6). In general, C K - M B activity usually peaked between 8 and 16 h postligation with peak levels ranging between 25.5-74.3 IU/1. The mean peak level of 49.6 + 21.4 IU/1 was ten times that seen in control animals and also occurred at 12 h as in the case of Tn-I (Fig. 7). Thereafter, C K - M B levels declined rapidly reaching control levels between 40 to 100 h postligation.

Infarct size Infarct sizes determined by histochemical staining of heart slices (see Materials and Methods) ranged from 3.4% to 37.8% of the left ventricle (2.3% to 27.4% of whole heart) with a mean of 15.7%. The relationship between C K - M B and Tn-I estimates of infarct size in individual animals was examined using both peak values and areas under the time-activity curves.

Troponin-I in Myocardial Infarction

Cumulative release of C K M B determined from the time/activity curve plotted several days post-infarction has been used as part of a complex calculation to determine infarct size [23, 26, 29]. However it has been shown [9, 22] that not only cumulative release but also both total area under the time-activity curve and peak values correlate well with infarct size. The latter two parameters were therefore selected in view of the complex nature of the T n - I release profile. These were correlated with infarct size measured histochemically. Both peak C K - M B and the area under the C K MB time-activity curve correlated well with histochemical infarct size (r = 0.87 and 0.9 respectively). In contrast, peak T n - I (r = 0.33) and T n - I time-activity ( r - --0.01) parameters showed poor correlation with histochemical infarct size. Comparison of peak C K - M B with that of peak Tn-! gave a correlation o f r = 0.52. Discussion The previously established radioimmunoassay [4] for cardiac Tn-I showed some limitations with regard to sensitivity and assay time which were largely overcome with the current ELISA. Some differences in sensitivity could have arisen due to the different species (i.e. canine compared with human) under investigation and the antisera sources involved. However preliminary observations indicate that the ELISA gives comparable results when used to measure human cardiac T n - I levels (B. Cummins and P. Cummins, unpublished observations). The lower limit of detection of the ELISA of 4 ng cardiac T n - I / m l serum allowed measurement of normal circulating levels in the dog of 15 ng/ml (range 9.3-21.5). This compares with an estimated mean of 11 ng/ml in man by radioimmunoassay [4] although due to the reduced sensitivity of 10 ng/ml in the latter it was not possible to measure normal circulating h u m a n levels with precision. At the maximum levels (200 ng/ml) detected in the current study, isolated whole cardiac troponin-I is completely soluble in normal serum. However this would not exclude the possibility of binding to other components of the troponin complex, the circulating levels of which have not to date been measured. The upper working limit for undi-

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luted serum in the ELISA was 200 ng/ml compared with 1000 ng/ml for the radioimmunoassay although dilution allowed assay of excess Tn-I levels. A further advantage of the ELISA over the radioimmunoassay was the reduced assay time of 5 to 6 h compared with 24 to 36 h. The ELISA was for all practical purposes specific for the cardiac T n - I isotype. Surprisingly, although a high degree of sequence homology likely exists between skeletal and cardiac canine isotypes [36], virtually all antisera raised to the cardiac isotype showed a relatively low cross reactivity with mixed skeletal isotypes. Although this could reflect a higher degree of primary sequence heterogeneity in the canine when compared with rabbit isotypes, this low antisera cross reactivity has been observed previously by us in other mammalian species. The more likely explanation for low cross reactivity is that antigenic determinants are concentrated on heterogeneous isotype sequences and/or on the unique N-terminal cardiac peptide. The low skeletal cross-reactivity was also supported by the relatively low change in antisera dilutions ( 1 : 8 0 0 0 0 compared with 1:50000) when comparing pre- and postskeletal Tn-I adsorbed anti cardiac Tn-I sera. More recently, we have noticed that a high proportion of monoclonal antibodies raised against cardiac Tn-I isotypes in different species are cardiac specific (but relatively species non-specific) failing to react with skeletal isotypes. This has important significance in terms of wider diagnostic assay development and suggests that cardiac immunochemical specificity is easily achieved with troponin-I. Examination of other contractile protein isotypes which have been employed in diagnostic immunoassays does not indicate the same potential for high cardiac specificity. Polyclonal antisera to cardiac myosin light chain subunits display cross reactivities ranging from 6.8% to 100% and 3% to 20% when tested against myosin light chains from human [15, 33, 39] and canine [18, 21] skeletal muscle respectively. Even the use of a combination of two monoclonal antibodies to different myosin light chain epitopes in sandwich immunoassay formats still gives a detectable skeletal crossreactivity of 3% to 4.3% [14, 28]. Monoclonal

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antibodies to cardiac myosin heavy chains show high cross reactivity with either the fast or slow skeletal isotypes depending on whether alpha or beta cardiac heavy chain isotype reacting monoclonals are employed [19]. In the case of tropomyosin, the cardiac and fast skeletal alpha isotypes are identical so that immunoassays for cardiovascular diagnostic purposes have limited potential [7]. In the present study the specificity of the ELISA for cardiac and not skeletal Tn-I was demonstrated by the lack of any detectable Tn-I release in the sham operated animals even though a considerable degree of skeletal muscle trauma and necrosis resulted from the thoracotomy. This confirms comparable findings clinically in which skeletal muscle trauma of varying severity fails to cause elevations of Tn-I in a cardiac assay [4]. Although problems were initially encountered with the use of C K - M B isoenzyme as a comparable cardiac specific marker in the canine model these were overcome using the fractionation procedure outlined in the Materials and Methods. The problems were two-fold, namely the previously reported very low levels of C K - M B in the canine myocardium of less than 2% of total [27, 30] compared with an average of 40% in man [38] but also the unexpected presence of low but detectable levels of C K B B isoenzyme in both normal cardiac and skeletal muscle. This was likely the source of the CK-BB present in normal canine serum. Although CK-BB is present in man to varying degrees in other organs (gastrointestinal tract > kidney > lungs > liver > spleen) [30] there is no previous evidence for the presence of CK-BB in human or canine cardiac or skeletal muscle. These complex distributions emphasize the importance of confirming the fractionation of CK isoenzymes using ion exchange kits by electrophoretic analysis. Notwithstanding these problems it was clear that C K M B isoenzyme was present in canine cardiac but not skeletal muscle. The time course of cardiac Tn-I release post experimental infarction in the dog was similar to that observed after clinical infarction [4]. Tn-I levels were detectable between 4 to 6 h after ligation, identical to that post infarction in man, with a slightly earlier peak time of

1 2 h (range 10 to 16h) compared to 18h (range 15 to 24 h). This may reflect a more gradual development of necrosis in the clinical situation consequent upon longer term development of coronary occlusion. Peak cardiac Tn-I levels were elevated approximately 10 to 11-fold in man compared with 6-fold in the canine model. Cardiac Tn-I levels were elevated above normal for upto 200 h postligation which was similar to that (8 days) seen in man [4]. The prolonged release of contractile proteins post-infarction has been observed in previous studies on myosin light [15, 20] and heavy chain subunits [19] and tropomyosin [7]. It is possible that this reflects release from two intracellular pools, one free in the cytoplasm and rapidly released and the second due to slow degradation ofmyofibrils. The Tn-I release profile did not readily lend itself to mathematical analysis. Determination of the cumulative release of Tn-I in a manner comparable to that previously established for C K M B isoenzyme [23, 26, 29] requires calculation of the disappearance rate from the downslope of the time-activity curve. The Tn-I profile did not allow simple comparison of cumulative Tn-I release as an estimate of infarct size with that directly determined by histochemical measurement. While good correlation existed in the current study between histochemical infarct size and C K - M B criteria when expressed either as peak values or areas under time activity curves, there was poor correlation between similar Tn-I criteria. The reasons for this are not clear but it is possible that while these aherative criteria are suitable for C K - M B they are unsatisfactory for the more complex Tn-I profile. It was noted that a somewhat better correlation existed between peak Tn-I and peak C K - M B levels which has also been observed clinically post-infarction [4] although in the latter study the correlation was higher. Further studies on the clearance rates from the circulation of injected radiolabelled cardiac Tn-I and C K - M B isoenzyme may help to identify these differences. In conclusion, the findings of the present study indicate that the ELISA will prove to be a suitable and improved alternative to radioimmunoassay for measuring circulating levels of cardiac troponin-I. However, while

Troponln-I in Myocardial Infarction

both cardiac Tn-I and C K - M B isoenzyme were shown to be specific indicators of myocardial necrosis in the canine model, only the release of C K - M B showed satisfactory correlation with histochemical infarct size.

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Acknowledgements This research was generously supported by a grant from Bayer U.K. Ltd. We would like to express particular thanks to Dr Brian Alps for his helpful advice on the surgical procedures.

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