Toxicologic Pathology, 37: 617-628, 2009

Copyright # 2009 by The Author(s)

ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1177/0192623309339502

Qualification of Cardiac Troponin I Concentration in Mouse

Serum Using Isoproterenol and Implementation in Pharmacology
Studies to Accelerate Drug Development
STEVEN K. ENGLE,1 WILLIAM H. JORDAN,1 MICHAEL L. PRITT,1 ALAN Y. CHIANG,1 MYRTLE A. DAVIS,1,2
JOHN L. ZIMMERMANN,1,3 DANIEL G. RUDMANN,1 KATHLEEN M. HEINZ-TAHENY,1 ARMANDO R. IRIZARRY,1 YUMI YAMAMOTO,1
DAVID MENDEL,1 A. ERIC SCHULTZE,1 PAUL D. CORNWELL,1 AND DAVID E. WATSON1
1
Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, Indiana, 46285, USA
2
National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 20892, USA
3
Vet Path Services, Inc., Mason, Ohio, 45040, USA
ABSTRACT

Cardiac troponin I is a useful biomarker of myocardial injury, but its use in mice and application to early drug discovery are not well described. The
authors investigated the relationship between cTnI concentration in serum and histologic lesions in heart tissue from mice treated with isoproterenol
(ISO). Cardiac TnI concentrations in serum increased in a dose-dependant manner and remained increased twenty-four to forty-eight hours after a single
administration of isoproterenol. Increased cTnI concentration was of greater magnitude and longer duration than increased fatty acid binding protein 3
concentration, aspartate aminotransferase activity, and creatine kinase activity in serum. Isoproterenol-induced increases in cTnI concentrations were
both greater and more sustained in BALB/c than in CD1 mice and correlated with incidence and severity of lesions observed in heart sections from both
strains. In drug development studies in BALB/c mice with novel kinase inhibitors, cTnI concentration was a reliable stand-alone biomarker of cardiac
injury and was used in combination with measurements of in vivo target inhibition to demonstrate an off-target contribution to cardiotoxicity. Additional
attributes, including low cost and rapid turnaround time, made cTnI concentration in serum invaluable for detecting cardiotoxicity, exploring structure–
activity relationships, and prioritizing development of compounds with improved safety profiles early in drug discovery.

Keywords: troponin; biomarker; cardiac; necrosis; drug development; mouse.

INTRODUCTION compared to measurement of a serum-based biomarker. Electron

Cardiac toxicity is an unintended and undesirable consequence
of exposure to many drugs, including medications used to treat
cancer, retroviral infection, diabetes mellitus, chronic obstructive
pulmonary disease, inflammation, and psychoses (O’Brien
2006). Preclinical safety assessment in animals has demonstrated
high predictivity of human cardiac toxicities, with up to 80%
concordance between preclinical and clinical studies (Olson
et al. 2000). Histological observation of heart lesions provides
indisputable evidence of cardiac injury in drug development and
preclinical studies and is routinely utilized during safety assess-
ment. However, it is not commonly employed during pharmacol-
ogy studies because it is relatively time-consuming and expensive
Address correspondence to: Steven K. Engle or David E. Watson, Lilly
Corporate Center, Indianapolis, IN 46285, USA; phone: 317-655-2121 or
317-433-2155; e-mail: Englesk@lilly.com or Davewatson@lilly.com.
Financial Disclosure: The studies described in this article were funded by
Eli Lilly and Company. The authors have not declared any other conflicting
interests.
Abbreviations: AST, aspartate aminotransferase (also known as serum
glutamic oxaloacetic transaminase); CK, creatine kinase; CMC,
carboxymethylcellulose; cTnC, cardiac troponin calcium binding subunit;
cTnI, cardiac troponin inhibitory subunit (Tnni3); cTnT, cardiac troponin
tropomyosin binding subunit (Tnnt2); CV, coefficient of variation; Fabp3,
fatty acid binding protein 3; GenBank, Protein symbols shown in
parentheses.; ISO, isoproterenol; IVTI, in vivo target inhibition; LLQ, lower
limit of quantitation; sc, subcutaneous; ULQ, upper limit of quantitation.
microscopy, while more sensitive than histology, is even more
time-consuming and expensive. In contrast, analysis for serum-
based biomarkers is cost-effective and rapid. A sufficiently sen-
sitive and specific biomarker can be used as a stand-alone safety
endpoint to prioritize compounds at early stages in drug develop-
ment. Unfortunately, traditional indicators of cardiac injury, such
as aspartate aminotransferase (AST) and creatine kinase (CK)
activities in serum, have limited sensitivity and lack specificity
for cardiac injury.
Cardiac troponin I is a specific, sensitive, and quantitative
measure of cardiac injury in humans (Adams et al. 1993;
Babuin and Jaffe 2005) and veterinary species (O’Brien et al.
2006). Concentrations in serum are increased as a result of
direct myocardial injury, myocardial ischemia, ventricular
strain caused by disease (Fromm 2007), or drug-induced toxi-
city (Babuin and Jaffe 2005; Adamcova et al. 2007). Cardiac
TnI has thus become the biomarker of choice for monitoring
cardiac injury in humans (Collinson and Gaze 2007).
Cardiac TnI is a sarcomeric protein in the troponin complex
of cardiomyocytes. The complex consists of troponins I (cTnI),
the inhibitory subunit; T (cTnT), the tropomyosin binding
subunit; and C (cTnC), the calcium binding subunit. Upon
membrane disruption, intracellular cTnI leaks into the extra-
cellular environment as a monomer, part of a binary complex
with cTnC, or in a ternary complex of cTnT-cTnI-cTnC
(Wu et al. 1998). Cardiac troponin I is distinguished from

617
618 ENGLE ET AL.
skeletal muscle isoforms of TnI by a cardiac-specific N-
terminal sequence (Larue et al. 1992), which is the basis for its
diagnostic specificity for myocardial damage.
Release of cTnI from myocardium was proportional to the
size and extent of tissue injury in several animal models of car-
diotoxicity (O’Brien et al. 2006) and has been reported to cor-
relate well with systolic function (Adamcova et al. 2007).
Challenges remain with regard to preclinical applications,
including assessment of a suitable cutoff for irreversible myo-
cardial injury, optimal timing of sample collection (Adamcova
et al. 2005), relatively limited data sets for animals, and lack of
standardization between various immunoassays available to
measure cTnI concentration. Important differences exist in
species-specific cross-reactivity of the antibodies used in the
assays available for the measurement of cTnI concentrations
in laboratory rats, dogs, and monkeys, as well as the precision
and accuracy of the measurements (Apple et al. 2008). There is
little information available regarding the use of these same
assays in mice. These challenges contribute to the limited use
of cTnI in toxicology studies, even those involving compounds
with previously identified cardiotoxic potential. Furthermore,
strains of mice used in discovery biology studies often differ
from those used in toxicology studies, contributing to uncer-
tainty in the interpretation of toxicology measurements in
‘‘nonstandard’’ strains of rodent.
To extend the application of cTnI as a biomarker of cardiac
injury in preclinical species, we qualified cTnI concentration in
serum as a biomarker of cardiotoxicity in CD-1 and BALB/c
mice treated with a nonspecific b-adrenergic agonist, isoproter-
enol (ISO), a well-described model of cardiac necrosis (Zhang
et al. 2008). Mice, because they require relatively small
amounts of compound, are a preferred species for in vivo
assessment of target inhibition and efficacy. In these studies,
we explored the kinetics of alterations in cTnI concentrations
in dose response and time course studies, as well as the rela-
tionship of cTnI concentrations to fatty acid binding protein
3 (Fabp3) concentrations, CK and AST activities, and severity
of myocardial injury detected by histopathological examina-
tion. Fabp3, also known as heart-type fatty acid binding pro-
tein, is a marker of tissue injury (Pelsers, Hermens, and Glatz
2005) with reported utility as a biomarker of cardiac necrosis
(Ruzgar et al. 2006), but its use has not been described in mice.
CD-1 and BALB/c mice were chosen because of their common
use in toxicology and pharmacology studies, respectively. The
comparison of increased cTnI concentration in serum from
both strains with histology provides assurance that the results
of cTnI screening in BALB/c mice will be comparable to the
results of a toxicology study in CD-1 mice.
Cardiac TnI concentration in serum was then deployed as a
stand-alone safety marker to detect myocardial injury caused
by novel kinase inhibitors in early drug discovery studies using
BALB/c mice. In vivo target inhibition was assessed concur-
rently in these studies. This strategy resulted in the identification
of unacceptably cardiotoxic compounds very early in the discov-
ery process and enabled prioritization of compounds that had a
favorable balance between target inhibition (potency) and safety.
TOXICOLOGIC PATHOLOGY
METHODS
Animals
Animal protocols were approved by Eli Lilly and Com-
pany’s Institutional Animal Care and Use Committee. Female
CD-1 and BALB/c mice were obtained at nine to eleven weeks
of age from Charles River Laboratories (Wilmington, MA) or
Harlan Sprague Dawley, Inc. (Indianapolis, IN). Female mice
were chosen for consistency with pharmacology studies in drug
development, in which female BALB/c mice are used. Mice
were housed singly with access to food and water ad libitum
and were allowed to acclimate for at least one week to caging,
feeding, and watering conditions before any handling. Blood
was collected under isoflurane anesthesia via retro-orbital sinus
or abdominal vena cava. Mice were humanely euthanized
either by exsanguination under anesthesia followed by organ
removal or by carbon dioxide inhalation followed by cervical
dislocation.
Isoproterenol Studies
ISO (Sigma-Aldrich, St. Louis, MO) was dissolved in 0.9%
sodium chloride and administered in a single, sc administration
at 0.1, 1, 10, or 50 mg/kg to BALB/c and CD-1 mice. Control
mice were treated with 0.9% sodium chloride. Dose volume
was 5 ml/kg. In a series of seven studies, blood was collected
from anesthetized mice via abdominal vena cava 1, 4, 8, 24,
48, or 72 hours after injection and processed for serum. Hearts
were collected for histopathologic examination 24, 48, or 72
hours after injection.
Histopathologic Examinations in Isoproterenol Studies
Hearts were fixed for histological examination in 10% neu-
tral buffered formalin, bisected longitudinally through both
ventricles, processed, embedded (both halves) in paraffin, sec-
tioned at a thickness of 5 mm, and stained with hematoxylin and
eosin. Tissue sections were examined by light microscopy and
evaluated for necrosis and inflammation (cellular infiltration,
primarily mononuclear). Lesions were scored as follows: 0 ¼
normal, 1 ¼ minimal (very few, small foci affected, approx.
2 to 5 myofibers per histological section), 2 ¼ slight (identifi-
able at low magnification but estimated to involve < 8% of tis-
sue), 3 ¼ moderate (easily identifiable, 8% to 20% of tissue
affected), 4 ¼ marked (20% to 50% of tissue affected), and
5 ¼ severe (>50% affected). Interpretation was based on
changes in individuals within each group, but for presentation
purposes, necrosis severity scores were also averaged into a
group score. Following initial evaluations to establish the prin-
cipal changes, tissue sections were evaluated independently by
two board-certified veterinary pathologists without knowledge
of strain, time, or dose. The final scores represented the consen-
sus of both evaluators. Photomicrographs were extracted at 8X
and 20X from digital images taken on a ScanScope XT (Aperio
Technologies, Inc., Vista, CA) using a 20X objective.
Vol. 37, No. 5, 2009 IMPLEMENTATION OF cTnI IN MOUSE PHARMACOLOGY STUDIES
AST, CK, Fabp3, and cTnI
AST activity and total CK activity in serum were analyzed
using a Roche/Hitachi 917 automated analyzer (Indianapolis,
IN) according to the manufacturer’s protocol. Fabp3 concentra-
tion in serum was measured by a mouse/rat-specific ELISA
(Cat# HK403 Hycult Biotechnology, Uden, the Netherlands)
as previously described (Pritt et al. 2008). Cardiac TnI concen-
tration in serum was measured by a mouse-specific ELISA
(Cat# 2010-1-HS, Life Diagnostics, Inc., West Chester, PA).
Standard and sample volumes were reduced to 25 mL from the
manufacturer’s recommended 100 mL per well to conserve lim-
ited sample volume. Cardiac troponin I, purified from mouse
hearts by ion-exchange chromatography in conjunction with
calcium-dependent affinity chromatography on troponin-C
agarose (Cat# 7110, Life Diagnostics, Inc., West Chester,
PA), was used to construct a standard curve after dilution in
naı̈ve mouse serum provided with the kit. Concentration of pur-
ified cTnI for standards was verified by amino acid analysis as
previously described (van Wandelen and Cohen 1997). The
cTnI ELISA was qualified according to published criteria
(DeSilva et al. 2003). Specifically, the quantifiable range
was determined in mouse serum spiked with cTnI for which
total error (mean bias þ interrun covariance) was less than
30%. Samples from ISO studies with cTnI concentration >
10 ng/mL were diluted 1:2 and reanalyzed.
Baseline cTnI Concentration
Sera from vehicle-treated mice were assayed for cTnI con-
centration to assess baseline concentrations and what effect,
if any, dosing and blood collection routes had. Blood was col-
lected from anesthetized mice via retro-orbital sinus or abdom-
inal vena cava. Mice received a single sc administration of
vehicle (0.9% saline) in studies with ISO or 1 to 4 daily oral
vehicle (1% carboxymethylcellulose or 10% acacia) adminis-
trations in drug development studies.
Discrimination of On- versus Off-Target Toxicity in
Pharmacology Studies
In vivo target inhibition (IVTI) was measured 2 hours after a
single oral administration of a novel kinase inhibitor (dose
volume ¼ 10 mL/kg) at a range of dose levels in BALB/c mice
(n ¼ 3 to 4 mice per dose). IVTI was measured as phosphoryla-
tion status of a signal transduction protein in peripheral blood
mononuclear cells (PBMCs) using phospho-specific antibodies
and flow cytometry procedures similar to those previously
described (Krutzik and Nolan 2003; Krutzik et al. 2004). Com-
pound concentrations in plasma were measured using mass spec-
trometry. A nonlinear, sigmoidal fit of percentage IVTI versus
plasma compound concentrations (Prism, GraphPad Software,
Inc., La Jolla, CA) provided a quantitative relationship between
IVTI and plasma compound concentration that was used to inter-
pret serum cTnI data from toxicology studies (see below).
Cardiotoxicity was assessed using cTnI concentrations in
serum (n ¼ 3 to 12) 4 hours after the last of 4 daily, oral
619
administrations of a range of dose levels of each compound.
Limited blood volume in BALB/c mice precluded measurement
of percentage IVTI in toxicology studies. However, plasma com-
pound concentrations were measured at 1, 2, 4, 8, 16, and 24
hours (n ¼ 2 per time) after the first administration and used
to interpolate percentage IVTI from the previously established
relationship between plasma compound concentration and per-
centage IVTI (preceding paragraph). We then calculated per-
centage target inhibition  hour as area under the curve and
plotted these data against group mean cTnI concentration in
serum to assess the relationship between target inhibition and
cardiotoxicity.
Screening for Improved Margin of Safety in
Pharmacology Studies
In cardiotoxicity screening studies, female BALB/c mice
were treated orally with 150 mg/kg of novel kinase inhibitors
sharing a common target. Test articles were administered in a
1% carboxymethylcellulose (Thermo Fisher Scientific, Wal-
tham, MA) vehicle at 10 mL/kg. Blood was drawn from
anesthetized mice 4 hours after the second daily administration
via retro-orbital sinus from 5 mice per compound, processed
for serum, and assayed for cTnI concentration. Comparisons
were made across compounds using both IVTI and cTnI data.
A subset of compounds were advanced into more extensive
toxicology studies to test the ability of cTnI measurements in
screening studies to predict cardiotoxicity. In these studies,
female BALB/c mice were treated daily with a range of doses
for 4 days. Twenty-four hours after the fourth dose, hearts were
collected for histologic examination.
Measurement of cTnI and Fabp3 Concentrations in
Mouse Hearts
Left ventricles were collected at necropsy from mice and
homogenized as previously described (Pritt et al. 2008). Total
protein concentration was measured by Bradford assay (Pierce
cat# 23236, Thermo Fisher Scientific, Rockford, IL) of each tis-
sue supernatant sample. Cardiac TnI and Fabp3 concentrations
in supernatant were determined by ELISA (described above) and
expressed per microgram of total protein.
Statistical Analysis
Quantitative results for cTnI concentration were analyzed
using a three-factor analysis of variance. Factors in the model
included treatment, strain, time, and their interactions. Treat-
ment effect for each strain at each time point was evaluated
using a t-test at the .05 significance level. The mean difference
in cTnI concentration in strain (CD-1 vs. BALB/c mice) was
also tested at the .05 significance level at each time point.
Shapiro-Wilk’s test (Shapiro and Wilk 1965) for normality was
performed at the .01 significance level to detect outlying obser-
vations, and Levene’s test (Levene 1960) for homogeneity of
variance was performed at the .01 significance level to assist
in interpretation of the effects.
620
TABLE 1.—Cardiac TnI concentration in vehicle-treated mice.
cTnI concentration (ng/mL)a
<0.14 0.14–0.29 0.3–1 >1
Strain Route Number of administrations Number of mice in range
CD-1 scb 1 34 1 —d
BALB/c sc 1 51 — 1
poc 1 39 — 3
po 2 23 1 —
po 4 54 1 3
a
Blood sampling for cTnI analysis was done 2 to 4 hours after the second
administration.
b 10 mg/kg. The lowest concentration was 0.36 ng/ml (24 hours
sc vehicle was 0.9% saline.
c
po vehicle was 1% CMC or 10% acacia.
d
Zero observations in this range: —.
RESULTS
Qualification of cTnI Assay
The lower limit of quantitation (LLQ) of the ELISA used
for detection of purified mouse cTnI in mouse serum was
0.14 ng/mL; the upper limit of quantitation (ULQ) was 
10 ng/mL. Spike recovery samples were measured in triplicate for
4 to 5 runs at 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, and 0.14 ng/mL;
mean bias was 0.02, 0.27, –0.56, 0.35, 0.93, –0.23, –1.68, and
6.85%; interassay variability assessed as coefficient of
variation (%CV) was 4.3, 5.5, 4.0, 4.1, 6.8, 7.6, 9.0, and
10.5, respectively.
Baseline cTnI Concentration
Cardiac TnI concentration in serum was less than the LLQ
in 94.5% of vehicle-treated mice (Table 1), and no difference
in baseline cTnI concentration based on blood collection route
or vehicle was detectable. The few examples of quantifiable
cTnI concentration in vehicle-treated mice may have been due
to spontaneous myopathies or to the stress of handling. With
such low incidence of detectable serological cTnI concentra-
tions in vehicle-treated mice, most serum concentrations of
cTnI above the LLQ in treatment groups were likely to be
treatment-related.
Cardiac TnI Concentration in Isoproterenol Studies
In CD-1 mice, mean cTnI concentration in serum increased
in a dose-dependant manner from 0.1 mg/kg to 10 mg/kg ISO
during the first 24 hours after treatment (Figure 1A). The max-
imum mean cTnI concentration after treatment with 50 mg/kg
(5.9 ng/mL) was not significantly greater than the maximum
after treatment with 10 mg/kg. Thirty-five of 39 CD-1 mice
treated with ISO at 1 or 10 mg/kg had cTnI concentrations in
excess of LLQ in the first 24 hours after dosing. The lowest
measurable increase was 0.18 ng/mL (8 hours postdose), and
21 were in excess of 1 ng/mL. After administration of ISO at
1 and 10 mg/kg, maximum cTnI concentration in serum
occurred 1 to 4 hours after dosing, and in both cases, the lowest
ENGLE ET AL. TOXICOLOGIC PATHOLOGY
concentration (n ¼ 5) was 1.1 ng/mL. In CD-1 mice treated
with ISO at 0.1 or 50 mg/kg, cTnI concentration returned to
baseline by 24 hours. Cardiac TnI concentration in CD-1 mice
treated with ISO at 1 or 10 mg/kg returned to baseline by 48
hours postdose. Maximum concentration of cTnI occurred 1
hour postdose for CD-1 mice administered ISO at 0.1, 1, or
— 50 mg/kg and 1 to 4 hours postdose at 10 mg/kg.
— In BALB/c mice, mean cTnI concentration in serum
— increased in a dose-dependant manner after treatment with ISO
—
at 0.1 to 10 mg/kg ISO during the first 24 hours after treatment
1
(Figure 1B). Within 24 hours of dosing, cTnI concentration was
greater than LLQ in 42 of 43 animals treated with ISO at 1 or
postdose), and 37 were greater than 1 ng/ml. In BALB/c mice
treated with ISO at 0.001 and 0.01 mg/kg, cTnI was below
LLQ at both times tested (4 and 24 hr). Maximum concentra-
tion of cTnI occurred 4 hours postdose in BALB/c mice admi-
nistered ISO at 0.1 and 10 mg/kg and 4 to 24 hours postdose
at 1 mg/kg; the lowest concentration after 1 mg/kg was
0.86 ng/ml and occurred 4 hours postdose. Cardiac TnI con-
centration in serum from BALB/c mice treated with ISO at 1
or 10 mg/kg returned to baseline by 72 hours postdose.
BALB/c mice were not treated with ISO at 50 mg/kg.
Histological Observations in Hearts
Isoproterenol treatment-related changes in hearts included
dose-, time-, and strain-dependent incidence and severity of
myocardial necrosis and inflammation. Aside from one section
at 48 hours (n ¼ 5) with minimal inflammation, no important
changes occurred in CD-1 mice at 0.1 mg/kg (not tabulated;
hearts were not collected from BALB/c mice given 0.1 mg/
kg). Changes were poorly dose-responsive at 1 and 10 mg/kg
in both strains (Figure 2). Evidence for active myocardial
necrosis and inflammation peaked at 48 and 72 hours, respec-
tively, and were generally less prominent in CD-1 than BALB/
c mice (Figure 3A-B). Lesion severity ranged from minimal
(grade 1), represented by loss of striations (coagulative necro-
sis) in a few myofibers, to moderate (grade 3), which affected
multiple clusters of myofibers (Figure 4; additional details of
grading scheme in Methods section). Necrosis and inflamma-
tion occurred most frequently near the lumen of the left ventri-
cle, especially near the apex, but were found in any portion of
the left ventricle and rarely in other portions of the heart.
Vacuolation was a minor feature and likely represented a com-
bination of a degenerative change and evidence for edema as a
component of inflammation. Inflammation was characterized
by increased numbers of mononuclear leukocytes, many of
which were macrophages. Increased cellularity was likely
enhanced by early regenerative activities of cardiac myocytes.
Mean Fabp3 Concentration in Isoproterenol Studies
Mean Fabp3 concentration in serum was measured in mice
treated with vehicle (saline) and ISO at 10 mg/kg. Fabp3 con-
centration was below the lower limit of quantitation (1 ng/mL)
in 13 out of 15 vehicle-treated CD-1 mice. Fabp3 concentration
Vol. 37, No. 5, 2009 IMPLEMENTATION OF cTnI IN MOUSE PHARMACOLOGY STUDIES 621

FIGURE 1.—Cardiac TnI concentration in serum from isoproterenol (ISO)–treated mice. Dose response and time course studies after a single sub-
cutaneous administration of vehicle (saline) or ISO at 10, 1.0, or 0.1 mg/kg in (A) CD1 and (B) BALB/c mice. Data points and bars represent mean
+ SEM (n ¼ 3–10) cTnI concentration in serum. Blood was collected via retro-orbital sinus in isoflurane anesthetized mice. *Significantly
greater than control (p  .006).

FIGURE 2.—Necrosis and inflammation in histologic sections of hearts from isoproterenol studies. Histopathologic data from heart sections from
CD1 (open triangles) and BALB/c (closed triangles) mice at 24 (A, B), 48 (C, D), and 72 (E, F) hours after a single subcutaneous administration of
ISO. Necrosis was most severe 24 to 48 hours after treatment, and inflammation was most severe 48 to 72 hours after treatment.
622 ENGLE ET AL. TOXICOLOGIC PATHOLOGY

FIGURE 3.—Comparison of cardiac necrosis detected by histopathology and cTnI concentrations in serum. Severity of cardiac necrosis (0 ¼ normal,
1 ¼ minimal, 2 ¼ slight, 3 ¼ moderate) in histologic heart sections and cTnI concentration in serum from BALB/c (dashed lines) and CD1 (solid
lines) mice after treatment with 1 mg/kg (A, C) and 10 mg/kg (B, D) ISO. Data points represent mean + SEM (n ¼ 3–17). BALB/c significantly
different from CD1. *p ¼ .04, **p < .001.

was increased in CD-1 mice 4 hours after treatment with ISO
(p ¼ .05) and returned to control values by 8 hours postdose
(n ¼ 5; Figure 5C).
In contrast, Fabp3 concentration was above LLQ in 5 out of
6 vehicle-treated BALB/c mice. Mean Fabp3 concentration
was increased in BALB/c mice after treatment with ISO; max-
imum concentration occurred 1 hour after dosing and was sta-
tistically significant compared to control at 1 and 4 hours after
dosing (n ¼ 5, p < .003; Figure 5C). Eight hours after dosing,
mean Fabp3 concentration in serum had decreased but had not
returned to baseline.
AST and CK Activities in Isoproterenol Studies
AST and total CK activities, classical markers of cardiac
injury, were measured in the serum of mice treated with vehicle
or ISO at 10 mg/kg. Vehicle-treated control mice from all
CD-1 (n ¼ 28) and BALB/c (n ¼ 24-26) studies were used to
establish baselines for AST and CK. Baseline AST and CK
activities were comparable between strains. AST activities were
minimally increased at 1 and 8 hours after treatment with ISO at
10 mg/kg (n ¼ 4-5, p  .003) and were not different from base-
line values at 24 hours postdose (Figure 5E) in CD1 mice. Anal-
yses of AST and CK activities in BALB/c mice were
compromised by hemolysis, which is known to cause positive
interference. No significant alterations in the activity of AST
were measured at any time in BALB/c mice (n ¼ 3-8). Total
CK activity in ISO-treated CD-1 mice (n ¼ 4-5) was increased
1 hour postdose (p  .001) and was not different from baseline
values at 8 or 24 hours after dosing (Figure 5G). Activity of CK
in BALB/c mouse serum was markedly increased at 1 hour
postdose, suggestive of myocyte injury. At 8 and 24 hours post-
dose, individual mice had marked increases in the activity of
CK, but group mean was not different from baseline (n ¼ 1-5).
Strain Comparison for Biomarkers
Consistent with histological observations (Figure 3A-B),
concentrations of cTnI in serum were significantly greater in
BALB/c than CD-1 mice at 4, 24, and 48 hours and remained
increased above LLQ longer in BALB/c (48 hours) than CD-1
mice (24 hours) after treatment with ISO at 1 and 10 mg/kg
(Figure 3C-D). Increases in Fabp3 concentration compared to
control groups were both greater and more sustained in
BALB/c mice than CD-1 mice (Figure 5D) but were not as
great or sustained as increases in cTnI concentration in either
strain (Figure 5B). There was no apparent strain difference in
increases of AST or CK activities, and both were of lesser mag-
nitude than changes in cTnI (Figure 5F, H).
Vol. 37, No. 5, 2009 IMPLEMENTATION OF cTnI IN MOUSE PHARMACOLOGY STUDIES 623

FIGURE 4.—Distribution and microscopic features of myocardial changes in isoproterenol studies. Photomicrographs of H&E stained sections of
mouse hearts including a slightly affected CD-1 mouse (A, C) and a moderately affected BALB/c mouse (B, D) 48 hours after treatment with
1 mg/kg ISO. In the left ventricle (LV), multiple foci of pallor (arrows) and increased cellularity were apparent at low (8X) magnification (A,
B). Changes were rarely found in the right ventricle (RV). Normal myocardium (NM) was interspersed with foci of coagulative necrosis (arrows)
and increased cellularity at both low and high (20X, C, D) magnification. Consistent with greater cTnI values in the BALB/c mouse, coagulative
necrosis was more severe in that strain than in the CD-1 mouse.

Cardiac TnI and Fabp3 Concentrations in Left Ventricles
of BALB/c and CD-1 Mice
Concentrations of cTnI and Fabp3 in supernatants from
homogenized left ventricles of BALB/c mice (2.5 + 0.3 and
13.4 + 0.2 ng/mg total protein, n ¼ 5) were not different from
those from CD-1 mice (2.6 + 0.5 and 14.6 + 1.6 ng/mg total
protein, n ¼ 5). Consequently, an inherent difference in tissue
concentration cannot account for either strain-based differ-
ences in the response of these biomarkers in serum from the
two strains or for the differences in baseline serological Fabp3
concentration.
Discrimination of On- versus Off-Target Toxicity in
Pharmacology Studies Using BALB/c Mice
To discriminate between on-target and off-target mechan-
isms of cardiotoxicity, we explored the quantitative relation-
ship between IVTI and serum cTnI in BALB/c mice treated
with kinase inhibitors sharing a common target. Due to limited
blood volume in BALB/c mice, independent studies were con-
ducted for IVTI and toxicological response (serum cTnI). To
bridge between these studies, we measured plasma compound
concentrations in both.
In the event cardiotoxicity were target-mediated, little varia-
bility should occur across compounds in plots of percentage
IVTI versus serum cTnI. If the cardiotoxicity had a significant
off-target component, significant variability would occur
across compounds in plots of percentage IVTI versus serum
cTnI. A clear difference was observed across compounds
(Figure 6), suggesting a significant off-target component to the
acute cardiotoxicity caused by these kinase inhibitors and jus-
tifying further exploration of structure-activity relationships to
retain pharmacological activity while minimizing compound-
related cardiotoxicity.
Screening for Improved Margin of Safety in
Pharmacology Studies
Mice treated with kinase inhibitors in screening studies dis-
played a range of cTnI concentrations in serum that were not
consistently related to target inhibition. Compounds were
binned into high risk, moderate risk, and low risk by consider-
ing both the IVTI and the serum cTnI data (Table 2). Increased
624 ENGLE ET AL. TOXICOLOGIC PATHOLOGY

FIGURE 5.—Comparison of biomarkers of cardiac necrosis measured in serum from isoproterenol studies. CD1 (white columns) and BALB/c
(black columns) mice treated with 10 mg/kg ISO. Bars represent mean values (+ SEM) for cTnI (A), Fabp3 (C), AST (E), and CK (G) in serum,
and fold change from control for cTnI (B), Fabp3 (D), AST (F) and CK (H). *p  .05 vs. control.

cTnI concentrations in sera from screening studies were predic- DISCUSSION

tive of cardiac necrosis after extended dosing (Table 3). Com-
Biomarkers of target organ toxicity can be used as stand-
pounds that caused cTnI concentrations in excess of 1 ng/mL
alone safety endpoints in discovery biology studies during
in serum from one or more mice after 2 days caused cardiac
early drug development. In these studies, cTnI concentration
lesions observable through histopathology after 4 days of
in serum was qualified in BALB/c and CD-1 mice and then
treatment.
Vol. 37, No. 5, 2009 IMPLEMENTATION OF cTnI IN MOUSE PHARMACOLOGY STUDIES 625

TABLE 2.—Cardiotoxicity screening in BALB/c mice treated

orally with novel kinase inhibitors.

Pharmacology cTnI Concentration (ng/mL)a

Compoundb % IVTIc <0.14 0.14–0.29 0.3–1 >1 Decision

d
1 –10 5 — — —
2 18 4 1 — —
3 31 1 1 2 1
4 52 3 — 2 —
5 54 3 — 2 —
6 58 5 — — —
7 64 — — — 5
8 65 — 1 1 3
9 67 3 1 — 1
10 76 — — — 5
11e 88 — — — 4
FIGURE 6.—Target inhibition and cTnI concentration in serum from 12 90 — 1 3 1
kinase inhibitor studies. Compound concentration in plasma was mea- 13e 91 1 1 — 2
sured 2 hours after a single administration of a novel kinase inhibitor, 14 91 3 1 — 1
and cTnI in serum was measured 4 hours after the fourth daily admin- 15 93 1 3 — 1
istration. Percent in vivo target inhibition (IVTI) was calculated from a 16 93 4 — 1 —
quantitative relationship established between compound concentration 17 94 1 — 4 —
in plasma and target inhibition in peripheral blood mononuclear cells 18 94 — — — 5
in pharmacology studies. Cardiac troponin I concentration in serum 19 95 2 2 1 —
20 96 2 1 1 1
increased with target inhibition after treatment with compounds 7 and
21 99 — — — 5
27, but not with compound 2, and to a lesser extent with compounds 3
22e >100 — — 1 3
and 12. Data for these compounds also appear in Tables 2 and 3. 23 >100 5 — — —
24e >100 — — — 4
25 >100 4 — 1 —
implemented as a biomarker of acute cardiac necrosis in paral-
26 >100 — — 2 3
lel with in vivo target inhibition studies in BALB/c mice. 27f >100 — — 3 2
Benefits of such a strategy included discrimination between
Problematic (high risk—cardiac necrosis expected) ¼&. Potential for safety margin
on- and off-target mechanisms of toxicity and prioritization may exist (medium risk) ¼&. Promising (low risk—cardiac necrosis not expected) ¼
of the development of compounds with improved safety pro- &.
files. This strategy proved to be an efficient and quantitative a
Blood sampling for cTnI analysis was done 4 hours after the second administration;
method of detecting cardiotoxicity after treatment with novel values are number of mice in range.
b
Compounds (see also Figure 6) were administered once daily for cardiotoxicity
kinase inhibitors in a drug discovery program. screening at 150 mg/kg for 2 days.
Cardiac TnI concentration in serum was a sensitive and c
In vivo target inhibition (IVTI) was measured 2 hours after a single administration at
quantitative marker of acute cardiac injury induced by treat- 10 mg/kg in pharmacology studies.
d
Zero observations in this range: —; n ¼ 5 mice per compound.
ment with ISO in both BALB/c and CD-1 mice. Increased e
Sample missing due to early death.
cTnI concentration in serum was progressive with dose of ISO f
cTnI sample 2 hours after 2 administrations at 100 mg/kg.
and occurred concurrently with cardiac necrosis, although
morphological changes detectable by histopathologic exami- in the medium-risk category demonstrated a dose margin in
nation plateaued between 1 and 10 mg/kg. A threshold con- which compound administration did not cause cardiac necrosis.
centration of cTnI in serum was identified, above which an All compounds that caused cTnI concentrations in the high-risk
animal was considered to have experienced adverse cardiac category caused cardiac necrosis in 4-day studies. Given the
injury. This was considered to be the lowest cTnI concentra- lack of uniformity in the assays available to measure cTnI, the
tion in any sample at the time point during which maximum threshold concentrations are likely to vary depending on which
group mean concentration occurred. In both strains, this assay is used.
occurred from 1 to 4 hours postdose and was 0.7 ng/mL Isoproterenol-induced increases in cTnI concentrations
(CD-1) and 0.9 ng/mL (BALB/c). were both greater and more sustained in BALB/c than in
In practice, we found that cTnI concentrations in serum CD-1 mice, consistent with increased incidence and severity
from BALB/c mice after treatment with a series of novel kinase of morphological changes (greater number of muscle fibers
inhibitors could be binned into low-risk (BQL-0.29 ng/mL), involved or greater total volume of injury). The increased
medium-risk (0.3-1.0 ng/mL), and high-risk (>1.0 ng/mL) duration was likely attributable to higher initial concentra-
categories. Compounds used in 2-day screening studies from tions of cTnI in serum, resulting from more extensive injury
which all measured cTnI concentrations fell into the low-risk in BALB/c mice. Differences in cTnI concentration in serum
category were not observed to cause cardiac necrosis in at the same dose across strains could not be explained by dif-
4-day studies. Compounds that produced cTnI concentrations ferent concentrations of cytoplasmic cTnI in the left
626
TABLE 3.—Cardiac necrosis in studies with kinase inhibitors
advanced into dose-escalation studies.
Compounda Dose (mg/kg) Incidence of Necrosisb
2 50
2 150
4 100
4 300
4c 1000
6 50
6 150
6 500
7c 30
7c 100
12 30
12 100
12 150
15 50
15 150
15 500
25 30
25 100
25c 300
a
Compounds were administered to female BALB/c mice once daily for 4 days (see
Table 2 for in vivo target inhibition).
b
Hearts were collected for histology 24 hours after the fourth dose and assessed for
any degree of necrosis.
c
Sample(s) missing due to early death.
ventricles. Greater cTnI concentration in serum from BALB/c
mice is likely attributable to a greater number of injured car-
diac muscle fibers.
The cause(s) for the strain differences in response to cardiac
injury induced by exposure to ISO was not determined. How-
ever, possible causes include desensitization of b-adrenergic
receptors (Perrino et al. 2005), strain-dependent differences
in receptor density, cardiac capacity for aerobic metabolism,
or adaptive responses (Faulx et al. 2005, 2007). Desensitization
may also account for the lack of dose-response in CD-1 mice
treated with greater than 10 mg/kg ISO.
Cardiac TnI concentration in serum was increased in CD-1
mice treated with 0.1 mg/kg ISO. These increases were
below the threshold described above and occurred without
histologic evidence of myocardial necrosis. Although small
areas of injury could have been missed in histological exam-
ination of a single tissue section, it seems more likely that
these relatively small increases in cTnI were indicative of
myocardial membrane leakage without cell death. Cells
releasing cTnI likely experienced mechanical stress related
to increased contractility, or a transient period of ischemia,
not lasting the 10 to 20 minutes required to cause cell death
(Fromm and Roberts 2001). Conceivably, this resulted in
temporary membrane disruption and leakage of intracellular
contents from the cytoplasm. Similar scenarios have been
observed in baboons and dogs in which short episodes (15
minutes) of myocardial ischemia were induced, leading to
significant increases in plasma activity of total CK as well
ENGLE ET AL. TOXICOLOGIC PATHOLOGY
as the more cardiac specific CK-MB, with no histological
evidence of cardiac necrosis (Heyndrickx et al. 1985; Vatner,
Heyndrickx, and Fallon 1986).
Approximately 2% to 4% of total cTnI exists free in the
0/12 cytosol of ventricular myocytes (Mair et al. 1996; Wu and Feng
0/12 1998). The rapid, forty- to eighty-fold increases in cTnI con-
0/3 centration in serum in the first 1 to 8 hours after ISO treatment,
0/3
1/1
followed by a gradual decline in concentration over the next 48
0/3 hours, suggested that cytosolic cTnI was released immediately
0/3 upon injury. Release of sarcomere-bound cTnI may have been
0/3 inhibited by the gradual process of myocyte cell death and may
5/9 not have reached the circulation due to phagocytosis by infil-
7/10
2/3
trating inflammatory cells. Clinical evidence of this has been
1/3 observed in patients undergoing coronary artery bypass grafts
2/3 in which cTnI concentration in serum increased in the first
0/3 20 minutes following reperfusion of the heart, while b-type
3/3 myosin heavy chain remained within reference intervals. In
3/3
0/3
excised atrial appendages from the same patients, 8% of total
0/3 cTnI content was found free in the cytosol, compared to less
0/2 than 0.1% of b-MHC, leading to the conclusion that the imme-
diate increase in circulating cTnI was from the free cytosolic
pool (Bleier et al. 1998).
We also compared cTnI changes with other markers of car-
diac injury. Increased Fabp3 concentration in serum suggested
cell membrane disruption, with considerable magnitude of
change in BALB/c mice. However, the duration of increased
Fabp3 concentration was relatively limited in comparison to
cTnI, and it is also found in skeletal muscle (Pritt et al.
2008), confounding specific detection of myocardial injury.
The differences in Fabp3 concentrations between strains, in
both control and treated mice, were remarkable and unex-
pected. They could not be explained by differences in Fabp3
concentrations in the ventricles. The small magnitude of
increases in Fabp3 in CD-1 mice were insufficient to determine
whether a difference in clearance rate of Fabp3 from blood
exists between mouse strains but in any case cannot explain the
large differences in serum concentrations.
Increased AST and CK activities also suggested myocardial
injury, but the magnitudes of change in these markers were rel-
atively small and the time for their detection short compared to
alterations in the concentrations of cTnI. These limitations,
coupled with a lack of specificity for myocardium, limit their
use as stand-alone cardiac biomarkers.
Recent evidence supports the use of cardiac troponin con-
centrations in serum, in conjunction with examination of histo-
logical sections of heart (Santucci et al. 2007) and
echocardiography (Kogan et al. 2007; Adamcova et al. 2007)
to explore the relationships of compound structure with phar-
macology and cardiotoxicity in mice. Our experience suggests
that cTnI can be used to reliably and efficiently identify
xenobiotic-induced myocardial membrane disruption and
necrosis without the need for more resource-intensive histolo-
gical and imaging-based examinations in short screening
studies.
Vol. 37, No. 5, 2009 IMPLEMENTATION OF cTnI IN MOUSE PHARMACOLOGY STUDIES
Screening for increased cTnI concentration in serum bene-
fited an early-stage kinase inhibitor drug discovery program.
Its use as a stand-alone cardiac safety endpoint gave insight
into each compound’s safety while assessing its pharmacol-
ogy using the same biological model, thus shortening cycle
time and reducing associated costs. Cardiac troponin I con-
centration provided clear evidence of cardiac toxicity as phar-
macology was being assessed, allowing prioritization of
compounds according to potency, bioavailability, and safety.
Using cTnI as a stand-alone biomarker in mice allowed for
higher throughput of compounds with less demand on
resources than required to synthesize compounds for toxicol-
ogy studies in larger species, such as rat. Without such screen-
ing, the lack of a relationship between target modulation and
cardiotoxicity would not have been as quickly evident and
compound selection for more resource intensive studies not
as well informed. Subsequent studies revealed potent com-
pounds with less cardiac liability that were advanced into
more extensive toxicology studies with a greater chance of
a favorable safety profile.
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