Abstract
The investigations aimed to evaluate the usefulness of cardiac troponins as biomarkers of acute myocardial injury in the rat. Serum from female Hanover Wistar rats treated with a single intraperitoneal (IP) injection of isoproterenol (ISO) was assayed for cardiac troponin I (cTnI) (ACS: 180SE, Bayer), cTnI (Immulite 2000, Diagnostic Products Corporation) and cardiac troponin T (cTnT) (Elecsys 2010, Roche). In a time-course study (50.0 mg/kg ISO), serum cTnI (ACS:180SE) and cTnT increased above control levels at 1 hour postdosing, peaking at 2 hours (cTnI, 4.30 μg/L; cTnT, 1.79 μg/L), and declined to baseline by 48 hours, with histologic cardiac lesions first seen at 4 hours postdosing. The Immulite 2000 assay gave minimal cTnI signals, indicating poor immunoreactivity towards rat cTnI. In a dose-response study (0.25 to 20.0 mg/kg ISO), there was a trend for increasing cTnI (ACS:180SE) values with increasing ISO dose levels at 2 hours postdosing. By 24 hours, cTnI levels returned to baseline although chronic cardiac myodegeneration was present. We conclude that serum cTnI and cTnT levels are sensitive and specific biomarkers for detecting ISO induced myocardial injury in the rat. Serum troponin values reflect the development of histopathologic lesions; however peak troponin levels precede maximal lesion severity.
Introduction
In man, serum levels of the cardiac troponins (cTn), particularly troponin I (cTnI) and troponin T (cTnT), are widely used in the detection of acute myocardial infarction and a range of other cardiac conditions (Mair, 1997; Christenson and Azzazy, 1998; Panteghini, 2004; Adamcova et al., 2005; Apple et al., 2005; Dybdahl et al., 2005). However, the use of serum cTnI and cTnT in the detection of cardiac injury in laboratory animal species is not as widespread (Herman et al., 1998; Bertsch et al., 1999; Bertinchant et al., 2000; Adamcova et al., 2005; Walker, 2006). Wallace et al. (2004) reviewed the usefulness of serum troponins as biomarkers of drug-induced cardiac toxicity in preclinical safety assessment studies as part of the role of the Expert Working Group (EWG) of the Nonclinical Studies Subcommittee (NCSS), which had been established by the US Food and Drug Administration, and which reported to the Advisory Committee for Pharmaceutical Sciences (ACPS).
In considering the case for advocating the increased use of cTn, the NCSS wished to improve the accuracy by which preclinical studies predict a clinical outcome with respect to potential adverse drug reactions, and also propose new biomarkers that would strengthen the interface between preclinical laboratory studies and clinical trials (Wallace et al., 2004). In addition, the EWG also set out the desired characteristics of the ideal serum/plasma biomarker for the detection of myocardial injury, and suggested that these were: specificity, sensitivity, predictability and robustness (Wallace et al., 2004). Nevertheless, the EWG also concluded that additional work was required for the further validation of the utility of cTn, and put forward a series of future goals, which included:
Evaluation of the kinetics of release and return to baseline (i.e. the diagnostic window) of cTn, and the correlation with histopathology, following the administration of compounds that cause distinct forms of cardiomyocyte injury.
Determination of whether there is a threshold for the increase in serum cTnI, and below which there is no substantial or sustained cardiomyocyte injury.
Establish whether there is a diagnostic advantage of measuring serum cTnI, or cTnT, or cTnI and cTnT, in the various forms of cardiomyocyte injury.
Establish whether the variation in the cTnI assay platforms will influence diagnostic sensitivity within and across laboratory animal species.
With the above proposals in mind, we have carried out a series of studies involving isoproterenol (ISO) induced cardiac injury in the female Hanover Wistar rat. ISO was the first pure β-adrenoceptor agonist to be synthesised (Sears and L̈otvall, 2005). The drug was developed in the 1940s and quickly became widely used for the relief of the symptoms of asthma (Waldeck, 2002). However, although ISO is a very potent β agonist, with almost no action on α-adrenoceptors, the drug does not distinguish between β1 and β2 receptors. As a result, ISO has significant extrapulmonary side effects, such as tachycardia, arrhythmias and palpitations, because the drug stimulates β1 receptors in the heart. ISO is a synthetic catecholamine and undergoes rapid metabolism, resulting in a very short duration of action (Dollery, 1998).
The drug is no longer used in the UK and USA for the relief of asthma symptoms (BNF, 2006). However, ISO has become widely used in toxicological studies as a model drug to induce cardiac muscle injury with myocardial ischemia and the formation of infarct-like lesions (Rona et al., 1959; Handforth, 1962; Judd and Wexler, 1974; Bleuel et al., 1995); the mechanism of toxicity is therefore closely related to the pharmacological action of the drug. In the rat, at high doses, ISO quickly stimulates β1 and β2 receptors in the heart, inducing an abnormally rapid heart rate (β1 activity) and a fall in blood pressure (β2 activity), resulting in cardiac tissue anoxia/hypoxia due to elevated oxygen demand and bringing about severe myocardial necrosis (Rona et al., 1959). These changes are associated with significant increases in both serum cTnI and cTnT (Bleuel et al., 1995; Bertinchant et al., 2000; Wallace et al., 2004). To induce cardiac lesions in the rat, ISO is generally administered by the subcutaneous, intraperitoneal or intravenous routes (Wexler and Kittinger, 1963; Leszkovszky and G′al, 1967; Kahn et al., 1969; Gryglewski et al., 1971; Benjamin et al., 1989; Bleuel et al., 1995; Mohan and Bloom, 1999; Inamoto et al., 2000; O’Brien et al., 2006) and elevations in the levels of serum cTn are generally evident within 4 hours of drug administration; the increases in serum cTn levels are closely correlated with the severity of myocardial injury (Wallace et al., 2004; Walker, 2006).
The purposes of the present investigations were: (1) to assess the linearity and precision of the cTnI assay using the ACS: 180SE (Bayer), together with the storage stability of the rat-specific immunoreactive cTnI signal; (2) to identify the onset of the release of cTn and correlate this timepoint with the nature and severity of the histopathologic lesion in the heart from 1 to 48 hours after the administration of ISO; (3) to identify the magnitude of the cTn signal with increasing dose levels of ISO over the dose range 0.25 to 20.0 mg/kg; (4) to correlate the magnitude and duration of the cTnI signal (ACS: 180SE; Bayer Healthcare Diagnostics) with the cTnT signal (Elecsys 2010; Roche Diagnostics) and also relate these 2 signals to the cTnI signal obtained with the DPC Immulite 2000 (Diagnostic Products Corporation); and, (5) to correlate the magnitude and duration of the cTnI signal with the more traditional biomarkers of cardiomyocyte injury, and in particular with both creatine kinase (CK) and lactate dehydrogenase (LD) isoenzymes. Preliminary reports of these findings have been published in abstract form (Chen et al., 2004; Brady et al., 2005).
Materials and Methods
Animals
Female Hanover Wistar rats (B and K Universal Ltd, Grimstone, Aldbrough, Hull, UK or Harlan UK Ltd, Bicester, Oxon, UK) were caged in groups of 3 to 6. Animals were bedded on wood shavings with diet (Rat and Mouse No. 1, SDS Ltd, Witham, Essex, UK) and mains drinking water ad libitum. A temperature of 19 to 21°C was maintained, with a relative humidity of 45% to 65% and a light: dark cycle of 12:12 h (lights on at 07.00 hours). Animals were allowed to acclimatise for at least 7 days before each experiment and were observed daily or more frequently for signs of ill health; body weights were determined at appropriate times. All animal procedures were carried out under local Ethical Committee guidelines and approval, and followed the Home Office (1989) “Code of Practice for the Housing and Care of Animals Used in Scientific Procedures.”
Administration of Isoproterenol
Isoproterenol (ISO; Sigma Chemical Co, Poole, Dorset, UK) (also known as isoprenaline in the UK) was dissolved in phosphate buffered saline (PBS) and administered by intraperitoneal (IP) injection; control animals were treated with PBS (vehicle) by the same route.
Autopsy Procedures and Sample Collection
Rats were sacrificed by IP injection of pentobarbitone sodium (Sagatal; Rĥone M′erieux Ltd., Harlow, Essex, UK) and blood was removed from the abdominal aorta and collected into serum separator tubes (Microtainer; Becton Dickinson and Co, Franklin Lakes, NJ, USA) and clotted at room temperature for 2 hours before centrifugation (6,000 rpm, 5 min). The serum was harvested and stored at −80°C prior to assessment.
Serum Clinical Chemistry
Serum cTnI levels were measured by automated immunochemiluminescence on the ACS: 180SE kit (Bayer Healthcare Diagnostics, Newbury, UK). The assay consists of antibodies directed against distinct sites of the cTnI protein. The capture antibody (solid phase) consists of a combination of 2 monoclonal antibodies; 80% of the solid phase is made up of a monoclonal antibody that is focused on the stable region of cTnI between amino acids 70–110, and 20% of the solid phase is made up of a second monoclonal antibody that is focussed on the P2 region, between amino acids 11–26. This last region is the cardiac-specific region (1–31) and was incorporated to assure capture of as many fragments of cTnI as possible. The “Lite reagent” tracer detection antibody is a polyclonal antibody which has been affinity purified against the P3 epitope and is made up of amino acids 27–40; this is the cardiac-specific area.
Serum cTnT levels were measured by automated immunochemiluminescence on the Elecsys 2010 (Roche Diagnostics, Lewes, UK). This assay used a biotinylated mouse monoclonal antibody for antigen capture (antibody M11.7 recognizing amino acid residues 136–147) linked to streptavidin-coated paramagnetic beads. The mouse monoclonal detection antibody (M7 recognizing amino acid residues 125–131) was labelled with ruthenium trisbypyridyl complex (Muller-Bardorff et al., 1997).
Additional assays of serum cTnI concentrations in selected experiments were also performed by automated immunochemiluminescence on the Immulite 2000 (Diagnostic Products Corporation, Llanberis, UK). This assay used a mouse monoclonal capture antibody and an alkaline phosphatase-conjugated polyclonal detection antibody, both directed towards epitopes betweeen amino acid residues 33–110 of cTnI (Scharnhost et al., 2002).
Total creatine kinase (CK), total lactate dehydrogenase (LD), alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamate dehydrogenase (GLD) and aldolase activities in serum were measured by automated spectrophotometric methods on the Advia 1650 (Bayer Healthcare Diagnostics, Newbury, UK) using commercial diagnostic kits. Electrophoresis of CK and LD isoenzymes was performed on the REP automated electrophoresis system (Helena Laboratories Corporation, Gateshead, UK).
Histopathological Examination
At autopsy, the heart was removed, weighed and immediately immersed in 10.5% phosphate-buffered formalin fixative. Following fixation, the hearts were trimmed using a standard pattern (Isaacs, 1998) to provide a transverse section through the middle of the ventricles and longitudinal sections of the apex and base of the heart. Tissues were processed routinely into paraffin and 3 μm sections cut and stained with hematoxylin and eosin (H&E). A single standard section from each animal was examined (blind review) microscopically by one operator and lesions graded as minimal (grade 1) (occasional individual myofibre injury), mild (grade 2) (multiple individual myofibre injury), moderate (grade 3) (larger focal to locally extensive areas of myofibre injury), or moderately severe (grade 4) (the majority of fibres in the myocardium affected).
Statistical Analysis and Data Presentation
ISO treated and control groups were compared using an unpaired 2-tailed Student’s
Experimental Design
Experiment 1: Characterization of the cTnI assay using the ACS: 180SE (Bayer)
Experiment 1a: Immunoreactivity and Linearity of the cTnI Response
Immunoreactivity and linearity of the cTnI response within the working range (0–50 μg/L) was assessed using (i) varying dilutions of rat cardiac homogenate prepared freshly in PBS; (ii) an experimentally-induced in vivo generated rat-specific cTnI in serum; (iii) purified commercial calibration materials (Hytest, Turku, Finland) prepared from rat cardiac homogenates. Dilutions of the material were prepared in species-specific cTnI negative serum.
Experiment 1b: Study on the Stability of cTnI in Serum
Female Hanover Wistar rats (n = 27; mean body weight 172.7 g; SD, 8.7 g; B and K Universal) were randomized into 2 groups; 22 animals were treated with ISO at 50.0 mg/kg by IP injection, and 5 (controls) were dosed with PBS (vehicle) by the same route. At 2 hours postdosing, rats were sacrificed by the IP injection of pentobarbitone sodium and blood removed from the abdominal aorta and collected into serum separator tubes that were maintained at room temperature (RT). At 2 hours after the autopsy, serum was prepared and assayed for cTnI. The mean level of cTnI, measured on the ACS: 180SE, for the ISO treated animals (n = 22) was 25.30 (SD: 13.28) μg/L, and for the control animals (n = 5), <0.03 (SD: 0.00) μg/L.
Additional measurement of cTnT was also performed to provide correlation data (reported later). The serum samples were then pooled to provide cTnI positive material; the level of cTnI in the pooled sample was 27.50 μg/L. The pooled sample was then divided into aliquots to investigate the immunoreactive cTnI stability in serum at RT, 4°C, −20°C, and −80°C.
Experiment 2: Time-Course Study (1 to 48 hours postdosing)
Female Hanover Wistar rats (n = 32; mean body weight 111.2 g; SD, 6.8 g; B and K Universal) were randomized into 8 groups of 4 animals each. ISO was given as a single IP injection at 50.0 mg/kg to 7 groups of animals, and one group (controls) received PBS (vehicle). Animals were autopsied at 0 hours (vehicle treated control), and at 1, 2, 4, 6, 12, 24, and 48 hours postdosing. Blood was removed for serum preparation and hearts were taken for histopathologic investigation. Serum cTnI levels were measured using the ACS: 180SE (Bayer) and with the Immulite 2000 (Diagnostic Products); cTnT was assayed using the Elecsys 2010 (Roche).
Experiment 3: Dose-Response Study (8.0 to 48.0 mg/kg Isoproterenol)
Female Hanover Wistar rats (n = 35; mean body weight 116.4 g; SD, 10.0 g; B and K Universal) were randomized into 7 groups of 5 animals each. ISO was administered by IP injection at 8.0, 16.0, 24.0, 32.0, 40.0 and 48.0 mg/kg; control animals were dosed with PBS (vehicle). Animals were autopsied at 5 hours postdosing. Blood was removed for the preparation of serum and hearts were taken for histologic study. As in Experiment 2, serum cTnI levels were assayed with the ACS: 180SE and with the DPC Immulite 2000; values for cTnT were determined using the Elecsys 2010.
Experiment 4: Dose-Response Study (0.25 to 20.0 mg/kg Isoproterenol)
Female Hanover Wistar rats (n = 108; mean body weight 124.1 g; SD, 15.4 g; Harlan UK) were randomised into 12 dose level groups of 9 animals each. ISO was administered by IP injection at 0.25, 0.5 and 1.0 mg/kg (part 1 dose level grouping); 2.0, 4.0 and 6.0 mg/kg (part 2 dose level grouping); and 8.0, 10.0 and 20.0 mg/kg (part 3 dose level grouping); each grouping of 3 ISO dose levels had a separate control group which was treated with PBS (vehicle) by the IP route. Of the 9 rats in each of the 12 part 1, 2, and 3 groupings, 5 were autopsied at 2 hours postdosing, and the remaining 4 animals were autopsied at 24 hours postdosing. At autopsy, blood was taken for the preparation of serum and hearts were placed in fixative for histologic study.
Serum was assayed for cTnI (ACS: 180SE) and for the levels of several serum enzymes conventionally used to assess cardiac toxicity, including LD (total activity) and LD isoenzymes (LD1, 2, 3, 4, and 5), CK (total activity) and CK isoenzymes (CKMB, CKBB and CKMM); also, ALT, AST, GLD and aldolase activities were measured. Histologic assessment of cardiac histopathology was carried out and involved the quantification of lesions on a scale of 0 to 4, and the mean lesion score at each ISO dose level was expressed as the myodegeneration score.
Results
Experiment 1a: Immunoreactivity and Linearity of the cTnI Response
Reproducible linear responses were found with all the immunoreactive cTnI materials tested over the dynamic range of the assay (0.03 to 50 μg/L). Serum cTnI levels in control/untreated rats were found to be below the lower limit of quantitation (<0.03 ug/L). Interassay coefficients of variation (CV: n = 50) were 11.1% (0.27 μg/L) and 6.3% (13.3, 33.2 μg/L).
Experiment 1b: Stability
Stability studies demonstrated that immunoreactive rat serum cTnI was stable for 24 hours (RT), 14 days (4°C) and up to 3 months (−20°C and −80°C). Additionally, no significant loss of immunoreactivity was observed following multiple freeze-thaw cycles (up to n = 5 cycles).
Experiment 2: Time-Course Study (1 to 48 Hours Dosing)
Levels of cTnI and cTnT, and the results from the histopathologic assessment of cardiac tissues, are presented in Table 1. The level of serum cTnI measured with the ACS: 180SE for vehicle treated (control) rats was <0.03 μg/L. ISO treated rats (n = 4) first showed a positive response at 1 hour post-ISO dosing. All 4 animals gave a positive response at 1 hour, with values ranging from 0.30 to 8.01 (mean 4.27, SD 3.86) μg/L. However, there was at this time some variability in the response. At 2 hours postdosing, values of cTnI measured with the ACS: 180SE ranged from 2.25 to 5.20 (mean 4.30, SD 1.38) μg/L. Levels of serum cTnI then showed a gradual decline from 4 to 24 hours postdosing; at 24 hours, individual values ranged from <0.03 to 0.33 (mean 0.11, SD 0.15) μg/L with only 2 of the 4 rats showing increases above baseline (control) values.
At 48 hours after dosing, cTnI values measured with the ACS: 180SE had fallen to control levels (<0.03 μg/L) in all 4 animals investigated. Therefore, from Table 1, it is seen that the peak concentration of cTnI was at 2 hours (
Levels of serum cTnT measured with the Elecsys 2010 showed a similarity in the pattern of responses to the results obtained for cTnI with the ACS: 180SE (Table 1); however, the fold responses for cTnT were generally greater than the fold increases for cTnI. Levels for cTnT were first positive at 1 hour postdosing with ISO; here, all 4 animals examined were positive, with values ranging from 0.13 to 2.86 (mean 1.54, SD 1.37) μg/L (mean control baseline levels were <0.01, SD 0.00 μg/L). As with cTnI levels, at 2 hours after ISO administration values of cTnT assayed with the Elecsys 2010 were higher than at 1 hour, the mean result being 1.79 μg/L (
As with cTnI, levels of cTnT then showed a general gradual decline from 4 to 24 hours postdosing, and at the 24- and 48-hour time points, only 4 of the 8 animals investigated showed a positive response above the baseline value of <0.01 μg/L. The release of cTnT paralleled the findings for cTnI, with cTnT values peaking at 2 hours postdosing; the mean level of cTnT at this time (1.79 μg/L) representing a 178-fold increase over the baseline control value. Similarly, as with cTnI, clear evidence of cTnT positivity was evident from 1 to 6 hours post-ISO dosing.
When serum cTnI concentrations were measured using the Immulite 2000 platform (Table 1), in contrast with the above findings for cTnI and cTnT, peak mean values were evident at 1 hour postdosing (0.48 μg/L), but this level represented less than a 1-fold increase over the control baseline value (0.37 μg/L). At other time points the results also indicated poor immunoreactivity and sensitivity (Table 1) with this assay.
Histopathologic examination of hearts from control and ISO treated rats showed that the earliest changes in the myocardium were evident at 4 hours after the administration of ISO (Figure 1) when a single animal of the 4 studied showed minimal (grade 1) myofibre eosinophilia (characterized by swelling and increased eosinophilia of cardiac myofibres). At 6 hours, 2 rats of the 4 examined demonstrated similar minimal to mild changes, with a third animal showing acute myodegeneration (characterized by swollen eosinophilic myofibres with a neutrophil infiltrate). At 12 hours dosing, 3 of the 4 rats investigated had minimal to mild acute degeneration, and at 24 hours and 48 hours, all animals showed mild to moderate chronic myodegeneration (characterized by a loss of cardiac myofibers and a mononuclear cell infiltration, graded as mild to moderate).
These results are presented in Table 1 and the relationship between the onset of histopathologic lesions and serum levels of cTnI and cTnT are illustrated in Figure 2. The first appearance of cardiac lesions, at 4 to 6 hours postdosing, is seen to be clearly demarcated from the times of onset of cTnI and cTnT positivity in the serum at 1 and 2 hours after the administration of ISO.
Experiment 3: Dose-Response Study (8.0 to 48.0 mg/kg Isoproterenol)
Results are presented in Table 2. It is seen that for cTnI levels assayed with the ACS: 180SE, from 8.0 to 48.0 mg/kg ISO, the mean fold increases of the 6 dose level groups ranged from 45.1 (at 16.0 mg/kg ISO) to 111.9 (at 48.0 mg/kg ISO); the mean of these 6 mean fold increase values was 71.05. However, there was no evidence of a dose-response relationship. For cTnI levels assayed with the Immulite, the mean fold increases of the 6 ISO dose level groups ranged from 0.3 (at 8.0, 16.0, and 40.0 mg/kg ISO) to 0.6 (at 32.0 and 48.0 mg/kg ISO); the mean of these 6 mean fold increases was 0.42. There was no evidence of a dose-response relationship. For cTnT levels assayed with the Elecsys 2010, the mean fold increases of the 6 ISO dose level groups ranged from 112.6 (at 16.0 mg/kg ISO) to 172.0 (at 48.0 mg/kg ISO); the mean of these 6 mean fold increases was 151.50. Again, a dose-response relationship was not evident.
These fold increases, in general terms, compared with the results obtained in Experiment 2 (above) and emphasize that results determined for cTnI with the ACS: 180SE, and for cTnT with the Elecsys 2010, show meaningful increases over the baseline control values. However, increases for cTnI obtained with the DPC Immulite, showed mean fold increases of 0.6 or less over the mean control value, suggesting low immunoreactivity.
An examination of the cTnI values for individual animals (data not shown) confirms the above findings. The 2 highest results for cTnI assayed with the ACS: 180SE were rat numbers 9 (8.0 mg/kg ISO) and 32 (48.0 mg/kg ISO), giving values of 9.67 μg/L (a 209-fold increase) and 14.47 μg/L (a 313-fold increase), respectively. With the Elecsys 2010, the values for cTnT for rat number 9 and 32 were 4.75 μg/L (a 474-fold increase) and 3.95 μg/L (a 394-fold increase), respectively; these 2 individual animals also had the highest cTnT levels of the 30 ISO treated rats. With the DPC Immulite, the highest values for cTnI were rat number 25 (32.0 mg/kg ISO) and 32 (48.0 mg/kg ISO), with values of 0.56 (a 1.3-fold increase) and 0.61 μg/L (a 1.5-fold increase), respectively.
Serum enzyme findings (data not shown) demonstrated that mean levels of ALT activity in ISO treated rats showed no clear or consistent increases above the vehicle treated control animals; similar negative results were found for mean AST levels. Results for mean levels of GLD, LD, CK and aldolase, in ISO treated animals, also showed no consistent dose-related increases above the mean baseline control values.
When the findings for serum enzyme levels in individual animals were considered, the only significant result appeared to be for rat number 9 given 8.0 mg/kg ISO, and rat number 32 given 48.0 mg/kg ISO. Rat 9 had a high cTnI value of 9.67 μg/L, and a cTnT value of 4.75 μg/L, which represented 209 and 474-fold increases, respectively. This individual animal had an AST level of 246 U/L (a fold increase of 1.67 above the baseline control mean value). Rat 32 demonstrated a cTnI level of 14.47 μg/L and a cTnT level of 3.95 μg/L, these being fold increases of 313 and 394, respectively. The AST activity of rat 32 was 249 U/L (a fold increase of 1.69 above the control mean value). The findings for rat 9 and rat 32 demonstrate clearly that the fold increases for cTnI and cTnT are very considerably greater than the fold increase of the more generally used serum enzyme AST, a conventional marker of cardiotoxicity.
The histologic assessment of hearts taken from ISO-treated rats at the autopsy at 5 hours postdosing (Table 2) demonstrated evidence of myofibre eosinophilia in a proportion of the animals from each ISO dose level group. This was graded as minimal (grade 1) in animals treated at 8.0 and 16.0 mg/kg ISO. The incidence of the lesions was increased at the higher ISO dose levels reaching a maximum in animals treated with ≥24.0 mg/kg. The increased incidence at ≥24.0 mg/kg was accompanied by an increase in severity grade in some animals (to mild myofibre eosinophilia) with a single animal (rat number 29) treated with 40.0 mg/kg ISO showing progression of the lesion type to mild acute myodegeneration. The lesions were predominantly localized to the left ventricular inner myocardium particularly affecting the apex of the heart and the papillary muscles. There appeared to be a trend for the magnitude of the histologic lesions to be associated with rats having higher cTnI values.
Experiment 4: Dose-Response Study (0.25 to 20.0 mg/kg Isoproterenol)
In this study, rats were dosed with ISO at lower dose levels than in Experiments 2 and 3. Selected serum clinical biochemistry results and histologic findings are presented in Table 3 (2 hours postdosing) and Table 4 (24 hours postdosing). At 2 hours post ISO dosing, serum cTnI positivity was evident at 0.25 mg/kg ISO, the lowest dose level of the drug administered; the mean value was 0.80 μg/L (P < 0.05), an increase of 25-fold above the mean control level of <0.03 μg/L (Table 3). Individual cTnI values for the 5 animals treated with ISO at 0.25 mg/kg were 0.23, 0.42, 0.67, 0.94, and 1.72 μg/L. This finding therefore clearly demonstrated that in this experiment the dose level of 0.25 mg/kg ISO was above “threshold” (i.e., a threshold dose was not identified). Above the dose level of 0.25 mg/kg ISO, there was a general trend for an increase in serum cTnI values with increasing dose levels of ISO.
The maximum positivity of cTnI, at 2 hours postdosing, was evident at 4.0, 6.0, and 8.0 mg/kg ISO with mean levels of 15.72 15.63, and 14.80 μg/L respectively, giving 261-fold, 259-fold, and 493-fold increases above the concurrent control values of 0.06, 0.06, and 0.03 μg/L, respectively. However, mean levels of cTnI at 10.0 and 20.0 mg/kg ISO were, at 2 hours postdosing, lower than at 4.0 and 6.0 mg/kg ISO, with mean values of 7.73 and 3.20 μg/L, respectively; the reasons for this result are unclear. Another feature of the cTnI values at these high dose levels of ISO was the variability of the response. For example, at 10.0 and 20.0 mg/kg ISO, 1 of the 5 animals at each dose level gave a result of <0.03 μg/L, the baseline (control) value. Indeed, baseline values were evident in 6 of the 45 animals treated with ISO at the 2 hour postdosing autopsy.
At 24 hours after the administration of ISO (Table 4), cTnI levels, in general, had fallen to baseline values at dose levels of 0.25 to 4.0 mg/kg ISO. However, at dose levels of 6.0, 8.0, and 10.0 mg/kg ISO there was still some evidence of mild positivity at 24 hours postdosing in some individual animals, with 5 out of a total of 12 animals treated at these ISO dose levels showing values of cTnI above baseline control values (<0.03 μg/L); this involved 2 out of 4 animals at 6.0 mg/kg ISO, 2 of 4 animals at 8.0 mg/kg, and 1 of 4 animals at 10.0 mg/kg. Nevertheless, at 20.0 mg/kg ISO at 24 hours postdosing, troponin positivity was still clearly evident in 3 animals treated at this dose level where cTnI values of 2.92, 6.33, and 15.43 μg/L, were observed. Nonetheless, this mean value of 6.20 μg/L, although not statistically significant, represented a 206-fold increase above the control baseline value of <0.03 μg/L. This finding of cTnI positivity at 24 hours postdosing at the high ISO dose level of 20.0 mg/kg was of interest, demonstrating that cTnI positivity and the magnitude of response was maintained at this time point.
Measurement of enzymes that have been conventionally used to assess injury in the heart, were examined at 2 hours post ISO dosing (Table 3). LD1 and LD2 isoenzymes were studied and results for both isoenzymes are given separately. These data are expressed as percentages of the total LD values, which are also presented (LD U/L) in Table 3. It is seen that statistically significant increased activities for LD1 and LD2 were evident at 0.25 mg/kg ISO. Indeed, the activity of LD1 was significantly increased at all dose levels of ISO at 2 hours postdosing (except at 0.5 mg/kg ISO), and activities of LD2 were significantly raised at dose levels of 2.0 mg/kg ISO and above. The maximum increase of LD1 was at 4.0 mg/kg ISO; here the activity was 7.8%, a 3.3-fold increase above the baseline control value of 1.8%.
For LD2, the maximum increase was also at 4.0 mg/kg ISO, and here there was a 4.6-fold increase above the baseline value. It is of interest to note that the results for LD1 and LD2, which show a pattern of response with a maximum peak at 4.0 mg/kg ISO, are, in general terms, similar to the pattern of response for cTnI, which also demonstrated a maximal peak at 4.0 mg/kg ISO. However, the fold increases for LD1 (3.3) and LD2 (4.6) at 4.0 mg/kg are considerably less than the fold increase (261) for cTnI at 2 hours postdosing with ISO.
It is also of interest to note that the mean results for LD2 (as % of the total LD values), at 2 hours after dosing with ISO, are equal to or above the mean results for LD1 (%) at all ISO dose levels except at 1.0 mg/kg ISO (Table 3). This pattern of change is also evident in the fold increases of LD1 and LD2: the fold increases for LD2 above baseline values at dose levels of 2.0 mg/kg ISO and above, are all higher that the fold increases for LD1; this appears to be a consistent observation.
At 24 hours post ISO dosing (Table 4), the mean activities of LD1 and LD2 had, in overall terms, returned to approximately normal levels; however, at 20.0 mg/kg ISO the levels of LD1 and LD2 remained above the baseline control values showing 2.3-fold increases in each case (
Another point of interest to emerge from the examination of the LD isoenzyme data at 24 hours post-ISO dosing (Table 4), is that where mean LD1 activities did show levels (as % or as fold increases) above baseline control values (NS or
For the isoenzyme CKMB at 2 hours post ISO dosing, the peak activity (as %) was at 4.0 mg/kg (Table 3), which again paralleled the findings for cTnI, LD1, and LD2; indeed, the increase for CKMB above control levels at 2.0 mg/kg ISO was 0.6-fold (
At 2 hours postdosing with ISO, levels of the serum enzymes ALT, AST, GLD, LD, CK, and aldolase showed, in general terms, no consistent, or dose-related increases (data not shown). However, in the case of AST, there was some evidence of small increases in activity in individual animals at the higher ISO dose levels. These small increases for AST were, for example, to 174, 195, 207, and 244 U/L and in these instances the levels of cTnI in these individual animals were also very high, being 15.07, 41.32, 17.40, and 28.42 μg/L, respectively. The mean concurrent control activity of AST was 119.0 U/L. For CK, at some higher ISO dose levels (2.0, 4.0 and 6.0 mg/kg) there was evidence of small but statistically significant decreases in the mean levels of activity at 2 hours postdosing (
The histopathologic assessment of cardiac lesions at 2 hours postdosing is set out in Table 3, with the changes quantified by severity grade on a scale of 0 to 4, and the mean (SD) of the grades expressed as the myodegeneration score. At 2 hours postdosing, 3 animals, 1 control and 1 each treated with 8.0 and 10.0 mg/kg ISO, showed minimal (grade 1) lesions characterized as chronic myodegeneration. Given the results from the time course study (Experiment 2), these observable lesions were considered to predate the start of the study and therefore represent background pathology. However, it is of interest to note that the 2 ISO treated individual animals had extremely high cTnI levels, 28.42 and 18.92 μg/L, respectively.
At 24 hours, post ISO dosing, all lesions seen in the hearts examined were characterized as chronic myodegeneration and the mean myodegeneration score showed a trend towards increasing severity with increasing dose levels of ISO (Table 4). At dose levels of 0.25 to 4.0 mg/kg ISO, the mean myodegeneration scores ranged from 1.0 to 2.3 (with
Discussion
In Experiment 1, the assay for cTnI using the Bayer ACS: 180SE platform was found to be immunoreactive and provide a linear response to rat-specific cardiac homogenate, purified standards and in vivo generated cTnI in rat serum over a dynamic range of 0.03 to 50 μg/L. Between batch precision of the assay (11.3%) was demonstrated to concentrations of 0.3 ug/L. Additionally the robustness of the assay was highlighted by exceptionally good stability of the immunoreactive cTnI signal when stored at 4°C or when frozen (−20°C and −80°C), and when samples were subjected to repetitive freeze-thaw cycles. Our findings in identifying the suitability of this assay to assess cTnI responses in the rat are supported by other workers (O’Brien et al., 2006) where the Bayer Centaur cTnI assay (which uses the same methodology and reagents as the ACS: 180SE) was evaluated.
In Experiment 2, it became evident that in this rat model of ISO-induced cardiac injury, there was a close association between the histopathologic assessment of acute/chronic myodegeneration and the serum levels of both cTnI and cTnT (Table 1), although there is clearly a temporal disconnect with maximal cTn responses preceding maximal severity of the histopathologic lesion observed. In Experiment 3 and 4 (Tables 2, 3, 4), serum cTnI and cTnT levels clearly demonstrated superiority over the previously used conventional/historical biomarkers of cardiac injury, such as AST, total LD, the LD isoenzymes LD1 and LD2, total CK, and the CK isoenzyme CKMB. Indeed, in the ISO treated rats (Tables 3, 4), AST, total LD, and total CK levels showed no clear or consistent dose-related increase above the vehicle treated control animals; there was however some evidence of small increases in AST activity in individual animals at higher ISO dose levels. Nevertheless, in general terms, the serum levels of the enzymes AST, total LD and total CK showed no evidence of having any useful diagnostic value in the evaluation of cardiac injury.
For the LD isoenzymes LD1 and LD2 in Experiment 4, the maximum fold increases at 2 hours postdosing (4.0 mg/kg ISO) were 3.3 and 4.6, respectively (Table 3); however, at this time point the minimum and maximum fold increases of cTnI in individual ISO treated rats were 25.3 and 492.5, respectively, demonstrating the clear superiority of cTnI as a diagnostically sensitive serum marker of cardiac injury. For the isoenzyme CKMB in Experiment 4, the maximum fold increase in ISO treated rats at 2 hours postdosing was 0.6 (4.0 mg/kg ISO) (Table 3); this poor result again confirms the clear advantage of cTnI as a serum marker of choice in ISO induced cardiac injury.
The increased relative activity of the isoenzymes LD1 and LD2 following cardiac injury, and the variation in the half-life of the 2 isoenzymes, has been discussed in several reports (Wolf et al., 1986; Preus et al., 1989; Ladi et al., 1990; Bertinchant et al., 2000; Walker, 2006). LD1 has been found to have the longest circulating half-life of the LD isoenzymes and relative shifts in the serum LD1/LD2 ratio have been shown to correlate with the severity and duration of cardiac injury (Wolf et al., 1986; Preus et al., 1988). At 2 hours postdosing in Experiment 4 (Table 3), activities of LD2 in ISO treated rats were generally equal to or higher than the activities of LD1. However, at 24 hours postdosing, LD1 activity was greater than LD2 (Table 4). These alterations in LD1 and LD2 isoenzyme levels over a 24-hour period have been referred to as the “LD1-LD2 flip,” and it has been suggested that these changes can be accounted for by LD1 having a longer half-life than LD2 (Walker, 2006).
It is seen (Table 1 and 2) that the magnitude of the increase in cTnT in the serum of ISO treated rats, above the baseline control values, is slightly greater than the magnitude of change in serum cTnI above baseline control values. For example, in Table 1, the maximum mean fold increase at 2 hours postdosing was 142.2 for cTnI and 178.0 for cTnT; in Table 2, the maximum mean fold increase at 5 hours postdosing was 111.9 for cTnI and 172.0 for cTnT with maximum fold increases for individual animals (data not shown) of 314 for cTnI and 474 for cTnT. However, in overall terms it is considered that the sensitivity of both biomarkers appears to be approximately similar in this particular experimental model. Furthermore, when serum samples from individual animals in Experiments 1, 2, and 3 were assayed for both cTnI and cTnT (n = 83), and the data re-analyzed and plotted with the line of best fit (Figure 3), cTnI and cTnT levels were shown to be closely correlated (R2 = 0.9202). This result would therefore indicate that either serum cTnI, or serum cTnT can be used as a suitable biomarker of ISO induced cardiac injury in the rat. However at cTnI levels below 0.3 μg/L with the ACS: 180SE, the precision of the assay was considered less sensitive compared to the cTnT assay (Table 1; Figure 3) but it is considered that interpretation of the experimental data was not compromised.
Nevertheless, these findings with cTnI measured with the ACS: 180SE (Bayer) and with cTnT measured with the Elecsys 2010 (Roche) are in clear contrast to the data on cTnI levels measured with the DPC Immulite (Diagnostic Products). Here the signal for cTnI was minimal; in Table 1 (Experiment 2) it is seen that the maximal mean fold increase in cTnI was 0.3 (at 1 hour postdosing), and in Experiment 3 the maximal fold increase for an individual animal was 1.5 (48.0 mg/kg ISO). These findings are supported by a recent report (O’Brien et al., 2006) where the dynamic range of the DPC Immulite assay was found to be approximately 1% of the Bayer Centaur assay in the detection of ISO induced cTnI release. These very low maximal fold increases determined with the DPC Immulite platform are considered to predominantly reflect differences in immunoreactivity and sensitivity, indicating the varying antibody epitope selectivities employed in the respective Bayer and DPC cTnI assays.
In the present investigations, serum levels of cTnI measured with the ACS: 180SE correlated closely with the onset of histopathologic findings (obtained from routine H&E stained sections) which were consistent with changes previously reported for this class of compounds (Van Vleet et al., 2002). Clear evidence of myofibre eosinophilia was seen in 1 of 4 animals (Experiment 2) at 4 hours post-ISO dosing (Table 1; Figure 1); acute myodegeneration in 1 of 4 rats was evident at 6 hours postdosing, and chronic myodegeneration in each of 4 animals was observed at 24 hours postdosing (Table 1; Figure 1).
In Experiment 3, minimal/mild myofibre eosinophilia was observed at 5 hours post ISO dosing, and in 1 animal (of a total of 30 treated with ISO) mild acute myodegeneration was also evident at this time point (Table 2). In Experiment 4 (Table 4), at 24 hours post ISO administration, chronic myodegeneration was seen in all drug treated animals, and there appeared to be evidence of increasing severity of the lesions with increasing dose level of ISO. These observations of histologic change at 24 hours were in concordance with the magnitude of troponin changes at 2 hours. It is considered that morphologic changes associated with cardiac injury could possibly be identified earlier than at 4–5 hours postdrug treatment with the use of transmission electron microscopy, or with special stains, or immuno/enzyme histochemistry.
A close examination of the data from the several experiments reported here shows that within a particular time-point group (Table 1), or ISO dose level group (Tables 2, 3, 4), serum cTnI (and cTnT) levels appear in certain cases to show considerable within-group variability, often in conjunction with a relatively large SD. However, this variability of cTnI/cTnT levels is also evident on the close examination of the published results of other workers (Bertinchant et al., 2000; O’Brien et al., 2006). The basis of this variability is unclear but it is probably multifactorial in origin. However, it is considered that the route of administration of ISO may be involved (i.e., the intraperitoneal route in the present studies), with resulting effects on the rate of drug absorption. Indeed, in more recent studies in our laboratories the drug has been given by subcutaneous administration and this appears to have reduced, to a degree, the amount of within-group variability. Nevertheless it is considered that the peak duration of cTnI release, or diagnostic window, following acute ISO induced cardiac injury was identified in these experiments and appropriate sampling for cTnI measurement with this particular mechanism of injury should be between 1–6 hours following dosing.
Another difference between the present investigations, and those recently reported by other workers, is that female rats were used. However, although there appears to be a current trend towards the use of male rats in studies on ISO induced cardiac toxicity (Ferrans et al., 1969; Lutmer and Wexler, 1971; Bleuel et al., 1995; Nakatsuji et al.,1997; Inamoto et al., 2000; Prabhu et al., 2006), nevertheless there still remain numerous examples of reports involving the use of female animals (Wexler et al., 1963; Kahn et al., 1969; Tang and Taylor, 1984; Hrdina and Kvetina, 1990; Bertsch et al., 1997; O’Brien et al., 2006).
At the present time, as the measurement of serum cTn in cardiac injury is a relatively new technique in toxicologic investigations, the place of the assay in the process of pre-clinical drug safety evaluation is unclear. It has been suggested that the measurement of cTn should be recommended for the identification of ongoing cardiomyocyte injury. However, it has also been proposed that cTn assays should not be advocated for inclusion in routine preclinical studies, but rather that the test would best be employed when further “follow-up” investigative examinations are required. Possibly, that is, when it has been established that some form of cardiac injury is present, or when it is known that acute tachycardia/arrhythmias have been induced. This would therefore suggest that the cTn assay may be used, not as a routine procedure, but rather as part of “reflective analysis” and a “case-by-case approach,” particularly if the time of onset of myocardial injury can be clearly defined. However the excellent sensitivity and specificity of cTn as a biomarker of cardiac injury will certainly encourage the consideration of the inclusion of cTn within the core screen, particularly with increased investigative use of this marker in sub-acute/chronic and chronic studies where the potential mode of cardiac injury may be quite different to the experimental model used in the present investigations.
A recent search of the literature has demonstrated that in studies involving cTn, the compound of frequent choice for the induction of cardiac lesions is ISO; also it would appear that ISO-induced changes in the heart are beginning to be characterized and reported in the literature, and related to serum levels of cTn. Nevertheless, this highlights the situation that in other types of cardiac injury, induced with other model compounds, cTn responses have not been well described and may have prolonged diagnostic windows for optimal measurement in comparison with ISO induced cardiac injury. This has become clear in studies on cardiac biomarker evaluation in our own laboratories where attention has now turned to examine different models of experimental injury with a series of other cardiotoxic agents.
In conclusion, and in agreement with the reports of Wallace et al. (2004), O’Brien et al. (2006) and Walker (2006), it is considered that in the present studies, cTn was confirmed as being a specific and sensitive biomarker for the detection of myocardial damage in the rat. On injury to the heart, cTnI is rapidly released, and in serum, cTnI is a robust marker and shows good stability in storage at 4°C, −20°C and −80°C. The duration of release of cTnI in the blood is also adequately long in acute injury. Increased levels of serum cTnI show an association with the development of cardiac lesions and precede maximal lesion severity in acute models of cardiac injury. Assays of cTnI in serum are rapid, specific, simple, accurate, relatively inexpensive, and easy to perform, although there is some evidence that technical improvements in the assay are required at low serum concentrations. The usefulness of cTnI as a biomarker is enhanced as it is not expressed in nontarget tissues and also because cTn bridges between preclinical and clinical studies.
Footnotes
Acknowledgments
We gratefully acknowledge the assistance of the technical staff at the School of Pharmacy for care of the animals and the technical support of the Clinical Pathology and Histology groups of GlaxoSmithKline, UK and the Clinical Pathology Laboratories of AstraZeneca, UK. SB acknowledges the support of GlaxoSmithKline, UK, and the School of Pharmacy.