Download Captopril Ameliorates Myocarditis in Acute

Captopril Ameliorates Myocarditis in Acute Experimental
Chagas Disease
Juan S. Leon, BA; Kegiang Wang, MD; David M. Engman, MD, PhD
Background—Captopril, an angiotensin-converting enzyme inhibitor, is commonly prescribed to patients with Chagas
heart disease (CHD). There are few human studies and no animal studies on the effects of captopril in CHD. We
investigated the effects of captopril on myocarditis and the host immune response to Trypanosoma cruzi in an
experimental model of acute CHD.
Methods and Results—A/J mice infected with Brazil strain of T cruzi developed acute myocarditis by day 21 after
infection, consisting of severe focal inflammation, necrosis, fibrosis, and T cruzi pseudocysts. Administration of
captopril (5 mg/L in the water) significantly reduced necrosis and fibrosis in infected mice. Increasing the captopril dose
also led to a decrease in inflammation. Captopril did not affect overall mortality but did delay death while having no
effect on parasitemia or cardiac parasite load. Treatment did not affect humoral immunity against T cruzi or cardiac
myosin (autoimmunity) but did decrease delayed-type hypersensitivity responses against both antigens. Interestingly,
increasing the dose of captopril induced mortality in infected mice in a dose-dependent manner. Mortality was
apparently not due to T cruzi because neither parasitemia nor cardiac parasitosis was affected. The combination of
captopril and infection may have impaired renal function because these mice had increased water consumption,
decreased body mass, and increased serum BUN/creatinine ratio.
Conclusions—Captopril ameliorates the myocarditis associated with acute T cruzi infection. (Circulation. 2003;107:22642269.)
Key Words: myocarditis 䡲 angiotensin 䡲 infection 䡲 collagen 䡲 myosin
C
hagas heart disease (CHD), caused by the protozoan
Trypanosoma cruzi, is a significant cause of morbidity
and mortality in South and Central America. Sixteen to
eighteen million people are infected, and 120 million people
are at risk of infection.1 CHD is a potentially fatal dilated
cardiomyopathy that develops in ⬇30% of T cruzi–infected
individuals. Treatment of clinical CHD is similar to that of
other cardiomyopathies, and includes sodium restriction and
treatment with digitalis, diuretics, and angiotensin-converting
enzyme (ACE) inhibitors, such as captopril and enalapril.2
Captopril binds to the peptide-binding pocket of ACE and
inhibits ACE’s catalytic production of angiotensin II; this
also promotes an increase in the level of bradykinin. By
interfering with the angiotensin II and bradykinin pathways,
captopril reduces systemic arterial pressure, peripheral vascular resistance, and cardiac filling pressure, and increases
cardiac output. Captopril is also an antiinflammatory agent,
acting through the immunomodulatory actions of angiotensin
II and the downstream effects of bradykinin (reviewed in
Godsel et al3). Together, the effects of captopril result in
reduced inflammation and fibrosis, improvement of cardiac
function, and enhanced survival in heart failure patients.
Despite routine administration of captopril to patients with
CHD, few studies have examined the effects of this drug on
these individuals. Captopril has been shown to improve
cardiac function with few side effects4,5 but has not been
found to reduce mortality in CHD.6 Of concern is the report
showing that in vitro administration of captopril enhances T
cruzi invasion of tissue culture cells by blocking the degradation of bradykinin.7 To date, however, there is no evidence
that captopril increases T cruzi parasitosis in humans. Captopril increases bacteremia in Pseudomonas aeruginosa–
infected mice8 and elevated bradykinin is associated with
bacteremia and mortality in Vibrio vulnificus–infected mice.9
Finally, prolonged captopril therapy for 6 months in a mouse
model of Coxsackie viral myocarditis reduced myocardial
fibrosis; however, mortality was increased for unknown
reasons.10 Whether this is relevant to T cruzi infection
remains to be determined.
To address these issues, we ascertained whether administration of captopril to T cruzi–infected mice would affect
myocarditis or the host immune response to infection. The
results provide compelling evidence that captopril ameliorates acute experimental CHD without affecting parasite load.
Received October 22, 2002; accepted January 21, 2003.
From the Departments of Pathology and Microbiology-Immunology and the Feinberg Cardiovascular Research Institute, Northwestern University
Feinberg School of Medicine, Chicago, Ill.
Correspondence to David M. Engman, Northwestern University Feinberg School of Medicine, Dept of Pathology, 303 E Chicago Ave, Ward 6-175,
Chicago, IL 60611. E-mail [email protected]
© 2003 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000062690.79456.D0
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Leon et al
Captopril Ameliorates Chagas Myocarditis
2265
Methods
Mice and T cruzi
Four- to six-week-old male A/J mice (Jackson Laboratories, Bar
Harbor, Maine) were housed under specific pathogen-free conditions. Mice were infected by intraperitoneal injection of 1⫻104
Brazil strain T cruzi trypomastigotes derived from infection of tissue
culture H9C2 rat myoblasts (American Type Culture Collection).
Parasitemia was measured from tailbleeds on a hemocytometer.
Uninfected controls received an intraperitoneal injection of Dulbecco’s Phosphate Buffered Saline (GibcoBRL) of equal volume. Mice
were anesthetized by a single intraperitoneal injection of 60 mg/kg
sodium pentobarbital for each experimental manipulation. The use
and care of mice were conducted in accordance with the guidelines
of the Center for Comparative Medicine (Northwestern University).
Captopril Regimen
Captopril was a gift from Dr Agostino Molteni (Northwestern
University, Chicago, Ill). Mice were given captopril at the indicated
concentrations in their drinking water from initial infection to day of
euthanasia. The captopril solution was changed 3 times a week and
prepared fresh from powder every time.
Preparation of Cardiac Myosin and
T cruzi Antigen
Cardiac myosin heavy chains were purified according to the method
of Shiverick et al,11 with modifications as described.12 T cruzi
antigen was prepared from T cruzi epimastigotes as described.12
Histopathology
Hearts were removed, rinsed with PBS, and fixed for 24 hours in
10% buffered formalin. Fixed hearts were embedded in paraffin,
sectioned, stained with hematoxylin-eosin or Masson’s trichrome,
and examined by light microscopy. Two sections were taken from
each heart, one including both atria and the other both ventricles.
Each section was examined for evidence of mononuclear and
polynuclear cellular inflammation, necrosis and mineralization, T
cruzi pseudocysts, and fibrosis and was assigned a histological score
between 0 (no involvement noted) to 4 (100% involvement), with 1,
2, and 3 representing 25%, 50%, and 75% involvement of the
histological section.13 Independent observers obtained substantial
(0.60 to 0.79) to almost perfect (0.80 to 1.00) agreement on their
scoring by weighted Kappa statistic14 as measured on a representative sample of 20 heart sections: overall agreement (0.82), inflammation (0.80), necrosis (0.96), pseudocysts (0.66), and fibrosis
(0.92).
Serological Analysis
Levels of cardiac myosin-specific and T cruzi–specific IgG were
determined by ELISA as described.12 End-point dilution titers for
total IgG were defined as the highest serum dilution that resulted in
an absorbance value (OD450) of two standard deviations above the
mean of a negative control (pooled sera from uninfected mice)
included in every plate.
Delayed-Type Hypersensitivity
Myosin- and T cruzi–specific delayed-type hypersensitivity (DTH)
was quantified using a standard ear swelling assay.12 Antigeninduced ear swelling was the result of mononuclear cell infiltration
and exhibited typical DTH kinetics (ie, minimal swelling at 4 hours,
maximal swelling at 24 to 48 hours after injection).
Clinical Chemistry
Serum levels of blood urea nitrogen (BUN), creatinine, and potassium were measured by the Center for Comparative Medicine
(Northwestern University) according to standard methods.
Figure 1. Histopathology scoring. Cardiac sections from
infected mice were stained with hematoxylin and eosin and
given inflammation scores of 0, 1, 2, 3, or 4 as described in
Methods. One section was stained with Masson’s trichrome and
given a fibrosis score of 3 (3M). Arrowheads indicate pseudocysts. Bar⫽50 ␮m.
Statistical Analyses
The statistical significance of DTH, blood chemistry, parasitemia, or
log-transformed (base 2) antibody titers were analyzed by 1-way
ANOVA followed by a 2-tailed t test and post hoc Bonferroni
analysis. Comparison of histological scores was analyzed by the
Pearson’s ␹2 test and post hoc Bonferroni analysis, and agreement of
blinded observer histopathology scores was analyzed by weighted
Kappa. Histopathological analysis was conducted on hearts of both
expired and euthanized mice unless otherwise stated. Significant
differences in mortality were analyzed by log rank test. Values of
P⬍0.05 were considered significant.
Results
Captopril Treatment of T cruzi–Infected A/J Mice
Decreases Cardiac Necrosis and Fibrosis
To investigate the effects of captopril on T cruzi–induced
myocarditis, we administered the drug to infected A/J mice in
their drinking water (5 mg/L). Twenty-five days after infection, analysis of cardiac histopathology (Figure 1) revealed a
significant decrease in necrosis and fibrosis in captopriltreated mice compared with untreated controls (Table 1). The
incidence of myocarditis (all treated and untreated mice
developed disease), body weight, heart weight, and heart
weight to body weight ratio (Table 2) were not affected by
2266
Circulation
May 6, 2003
TABLE 1. Captopril Reduces Necrosis and Fibrosis in Infected Mice
Scores
Captopril
5 mg/L
0
1
2
3
4
Total
⫹
1 (3)
12 (32)
18 (47)
7 (18)
0 (0)
38
⫺
1 (4)
13 (46)
10 (36)
4 (14)
0 (0)
28
⫹*
3 (8)
26 (68)
9 (24)
0 (0)
0 (0)
38
⫺
1 (4)
12 (43)
15 (54)
0 (0)
0 (0)
28
⫹
1 (3)
25 (66)
12 (32)
0 (0)
0 (0)
38
⫺
2 (7)
16 (57)
10 (36)
0 (0)
0 (0)
28
⫹*
3 (8)
22 (58)
12 (32)
1 (3)
0 (0)
38
⫺
0 (0)
8 (29)
15 (54)
4 (14)
1 (4)
28
Inflammation
Necrosis
Parasite
load
Fibrosis
Values are No. of mice (% mice). Captopril-treated, saline-injected mice
(n⫽10) and saline-injected mice (n⫽10) received scores of 0 for all parameters.
*P⬍0.05 compared with untreated infected mice.
captopril. Restricting analysis to mice surviving to 25 days
after infection did not change these results.
Captopril Administration Suppresses DTH but Not
Humoral Immune Responses in Infected Mice
We investigated whether the antiinflammatory properties of
captopril suppressed the immune response by assaying DTH
and antibody production to T cruzi and cardiac myosin
(autoimmunity). T cruzi DTH and myosin DTH were significantly lower in captopril-treated infected mice than in
untreated controls (Figure 2A). Interestingly, T-cell proliferative responses to T cruzi (not shown) and levels of T
cruzi–specific IgG and myosin-specific IgG were not affected
by captopril (Figure 2B). Both T cruzi–specific and myosinspecific IgM and IgG isotypes were also not affected by drug
treatment (data not shown).
Captopril Administration Does Not Affect
Parasitemia, Cardiac Parasite Tissue Load, or
Mortality in Infected Mice
We tested whether captopril affects host susceptibility to
infection by assessing mortality and parasite levels in treated
and untreated mice. Captopril (5 mg/L in the water) did not
affect mortality (Figure 3A), parasitemia (Figure 3B), or
cardiac parasitosis (Table 1). In 3 separate experiments,
Figure 2. Captopril reduces myosin-specific and T cruzi–specific
DTH but not humoral responses in infected mice. Four groups
included the following: 10 infected mice (Inf), 10 captopriltreated (5 mg/L) infected mice (Inf⫹Cap), 5 saline-injected mice
(Uninf), or 5 captopril-treated (5 mg/L) saline-injected mice. A,
Myosin-specific and T cruzi–specific DTH was measured by a
24-hour ear swelling assay as described in Methods. Error bars
indicate SEM. *P⬍0.01 compared with the infected group. B, At
21 days after infection, serum from each mouse was individually
tested for myosin-specific and T cruzi–specific IgG by ELISA.
Each data point represents the mean OD450 value of the mice in
each group, and the error bars indicate the SEM. Infected
groups had significantly (P⬍0.001) higher anti-myosin and
anti–T cruzi IgG that did their uninfected controls. Geometric
mean titers: anti–T cruzi–Inf⫽5250; Inf⫹Cap⫽4850; anti-MyosinInf⫽442; Inf⫹Cap⫽459.
captopril did, however, significantly delay death by 4 days
(P⬍0.05).
Increasing the Dose of Captopril Decreases
Cardiac Inflammation, Fibrosis, and Necrosis but
Increases Mortality in Infected Mice
We hypothesized that higher doses of captopril might further
reduce the severity of myocarditis. Administration of higher
doses of captopril up to 75 mg/L decreased cardiac inflam-
TABLE 2. Captopril Does Not Affect the Heart Weight or Body Weight of Infected Mice
Body, g
Heart, g
Ratio
⫺3
Infected (n⫽30)
21.4⫾0.5*
0.122⫾0.006*
5.8⫻10 ⫾3.0⫻10⫺4*
Infected⫹captopril (n⫽30)
21.5⫾0.4*
0.115⫾0.005
5.6⫻10⫺3⫾2.5⫻10⫺4*
Uninfected (n⫽15)
23.6⫾0.5
0.107⫾0.002
4.6⫻10⫺3⫾1.1⫻10⫺4
Uninfected⫹captopril (n⫽15)
23.8⫾0.6
0.106⫾0.003
4.4⫻10⫺3⫾1.6⫻10⫺4
Results are expressed as the mean of mice in that group⫾SEM from pooling 3 separate
experiments.
*P⬍0.05 compared with respective uninfected controls.
Leon et al
Captopril Ameliorates Chagas Myocarditis
2267
TABLE 3. Higher Doses of Captopril Reduce Inflammation,
Necrosis, and Fibrosis in Infected Mice
Scores
Captopril, mg/L
0
1
2
3
...
0 (0)
5
0 (0)
25*
50*
75*
4
Total
5 (25)
3 (15)
12 (60)
0 (0)
20
3 (15)
10 (50)
7 (35)
0 (0)
20
0 (0)
2 (10)
13 (65)
5 (25)
0 (0)
20
0 (0)
4 (20)
15 (75)
1 (5)
0 (0)
20
1 (5)
8 (40)
11 (55)
0 (0)
0 (0)
20
Inflammation
Necrosis
...
0 (0)
8 (40)
12 (60)
0 (0)
0 (0)
20
5*
10 (50)
10 (50)
0 (0)
0 (0)
0 (0)
20
25*
5 (25)
15 (75)
0 (0)
0 (0)
0 (0)
20
50*
5 (25)
14 (70)
1 (5)
0 (0)
0 (0)
20
75*
8 (40)
12 (60)
0 (0)
0 (0)
0 (0)
20
...
1 (5)
14 (70)
5 (25)
0 (0)
0 (0)
20
5
2 (10)
13 (65)
5 (25)
0 (0)
0 (0)
20
25
1 (5)
12 (60)
7 (35)
0 (0)
0 (0)
20
50
1 (5)
19 (95)
0 (0)
0 (0)
0 (0)
20
75*
16 (80)
4 (20)
0 (0)
0 (0)
0 (0)
20
Parasitemia
Fibrosis
Figure 3. Captopril has no effect on mortality or parasitemia of
infected mice. Groups of mice (see Figure 2) were monitored for
survival and parasitemia. A, Each data point represents the percentage of mice surviving at the particular time point from 3
separate experiments (n⫽30 for infected groups, n⫽15 for uninfected groups). B, Parasitemia was measured in each mouse
from each group at the indicated time points. Each data point
represents the average parasitemia of the group and error bars
are SEM.
mation, fibrosis, and necrosis in infected mice compared with
untreated controls at 21 days after infection (Table 3), but did
not further reduce the severity of myocarditis. Increasing the
dose of captopril decreased body weight and heart weight in
a dose-dependent manner, but had no significant effect on
heart weight to body weight ratio (data not shown). Cardiac
parasitosis was also not affected by increasing captopril dose,
except for mice treated at 75 mg/L (Table 3). Restricting the
analysis to mice that survived to 21 days after infection did
not change these results. Mice administered 75 mg/L of
captopril could not be analyzed in this manner because there
were not enough mice for statistical analysis (n⬍3) in 3
separate experiments. Surprisingly, increasing the captopril
dose also increased mortality in a dose-dependent manner
(data not shown). The increased mortality was not solely due
to direct captopril toxicity, however, because uninfected mice
treated with 75 mg/L of captopril had no morbidity or
mortality when examined out to 60 days after treatment.
Mortality was also not due to enhanced parasite load because
parasitemia (data not shown) and tissue parasitosis (Table 3)
were not affected by increasing the captopril dose. Further
analysis of these results revealed that increasing the dose of
...
0 (0)
5 (25)
13 (65)
2 (10)
0 (0)
20
5
1 (5)
12 (60)
7 (35)
0 (0)
0 (0)
20
25*
2 (10)
13 (65)
5 (25)
0 (0)
0 (0)
20
50*
3 (15)
16 (80)
1 (5)
0 (0)
0 (0)
20
75*
6 (30)
12 (60)
2 (10)
0 (0)
0 (0)
20
Values are No. of mice (% mice). Captopril-treated (75 mg/L), saline-injected
mice (n⫽5) and saline-injected mice (n⫽5) received scores of 0 for all
parameters.
*P⬍0.05 compared with untreated infected mice.
captopril led to increased water consumption and decreased
body mass (data not shown), suggesting that renal function
may be impaired in infected mice receiving higher doses of
captopril. Supporting this idea, infected mice treated with 75
mg/L captopril exhibited a significant increase in the serum
BUN/creatinine ratio compared with controls (Table 4).
Infection also significantly increased the BUN/creatinine
ratio compared with uninfected controls, whereas captopril
without infection had no effect.
Discussion
We investigated the effect of captopril treatment on the
outcome of T cruzi infection in mice. Captopril administration significantly decreased cardiac necrosis and fibrosis
without affecting mortality or host parasite burden. Captopril
decreased DTH to both T cruzi and cardiac myosin but had no
effect on T-cell proliferative responses to T cruzi or IgG
levels specific for either T cruzi or myosin. Finally, an
increase in the captopril dose decreased necrosis, fibrosis, and
inflammation, but also increased mortality.
These results are consistent with the few studies that
showed an amelioration of cardiac function in Chagas pa-
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Circulation
May 6, 2003
TABLE 4. Captopril Increases BUN, Creatinine, and BUN/Creatinine Ratio in
Infected Mice
Infected (n⫽10)
Infected⫹captopril (n⫽10)
BUN,
mg/dL
Creatinine,
mg/dL
BUN/Creatinine
Potassium,
mmol/L
25⫾1‡
0.34⫾0.04§
87⫾14‡
6.8⫾0.2
111⫾18*
0.62⫾0.11
181⫾34*
6.8⫾0.2
Uninfected (n⫽5)
26⫾2
0.48⫾0.11
66⫾13
6.4⫾0.2†
Uninfected⫹captopril (n⫽5)
31⫾3
0.42⫾0.02
75⫾8
6.9⫾0.1
Results are the mean of all serum measurements in mice at 15 days after infection in that
group⫾SEM.
*P⬍0.05 and †P⫽0.11 compared with Uninfected⫹captopril.
‡P⬍0.05 and §P⫽0.06 compared with Infected⫹captopril.
tients receiving captopril therapy4 – 6 and with results that
showed a reduction in myocarditis on captopril treatment in
other infectious myocarditides, including Coxsackie B315,16
and encephalomyocarditis virus infection.17–20 Captopril at 5
mg/L decreased fibrosis and necrosis but did not affect
inflammation. Increasing the captopril dose significantly
decreased inflammation (Table 3), in agreement with other
studies of infectious myocarditis.15–20 Overall mortality was
not affected by captopril at 5 mg/L (Figure 2A), although
death was delayed by 4 days. We hypothesize that the delay
in death is due to the antiinflammatory and antifibrotic effects
of captopril, which may protect the heart (this study and
Godsel et al3), lung,21 kidney,22 and other organs.
The potential concern that the antiinflammatory effects of
captopril may render the host susceptible to T cruzi infection
prompted us to investigate the effects of captopril on antigenspecific immunity. Captopril reduced T cruzi– and myosinspecific (autoimmune) DTH (Figure 2), but did not affect T
cruzi–specific T-cell proliferation or T cruzi and myosinspecific IgG levels. Both myosin- and ovalbumin-immunized
mice exhibit the same reduction in DTH and no effect on
T-cell proliferative and B-cell responses on captopril treatment (L.M. Godsel, PhD, unpublished data, 2002). Captopril
did not directly affect antigen-specific T- and B-cell function,
because neither in vitro nor in vivo administered captopril
directly affected antigen-specific T-cell proliferation or cytokine secretion (L.M. Godsel, PhD, unpublished data, 2002).
Thus, the decrease in DTH may not be due to a direct effect
on antigen-specific T-cell proliferation or cytokine secretion
but may be due to a reduction in other important aspects of
the inflammatory response, such as bradykinin production or
T-cell trafficking (L.M. Godsel, PhD, unpublished data,
2002).
Captopril does not seem to affect either the host response
or susceptibility to T cruzi. T cruzi parasitemia (Figure 3B)
and tissue load were not affected by captopril at any dose (not
shown and Table 3, respectively). These results are not
consistent with a previous report showing enhanced T cruzi
invasion of tissue culture cells on captopril treatment,7
perhaps because the host response is active at clearing T cruzi
despite enhanced susceptibility to invasion or because captopril concentrations in vivo are too low to enhance invasion.
Interestingly, captopril administration at 75 mg/L significantly reduced cardiac parasitosis, most likely because the
dead mice included in the analysis did not survive long
enough to have maximal cardiac parasitosis. Analysis of only
living mice was not possible because these mice did not
survive to a time point when histopathological analysis was
reproducible.
Increasing the captopril dose also enhanced mortality in a
dose-dependent manner, but did not enhance susceptibility to
T cruzi (preceding paragraph). Captopril administration has
also been shown to enhance mortality in Coxsackievirus–
infected mice through an unknown mechanism.10 We do not
know the precise cause of death in our mice. Mortality was
not due to hyperkalemia induced by captopril (Table 4). It is
possible that infection plus captopril administration impairs
renal function in these mice because captopril stimulates
water consumption,23 especially at higher doses, which in
turn increases drug intake. The increase in BUN/creatinine
ratios reflects impaired renal function, perhaps leading to
dehydration and decreased body mass.
Taken together, these results suggest that captopril can
reduce myocarditis and fibrosis without affecting host susceptibility to T cruzi infection. The mechanism of action of
captopril could involve suppression of angiotensin II levels,
enhancement of bradykinin levels, or a pharmacological
effect of captopril thiol group, among other mechanisms.
Antagonists of angiotensin II receptors reduced encephalomyocarditis virus–induced myocarditis.24 –26 Enhanced bradykinin levels and activation of nitric oxide and prostaglandins
by ACE inhibitors have been implicated in the reduction of
infarct size,27 hypertrophy,28 and reduced collagen gene
expression.29 Captopril’s cardioprotective effect may also be
due to its upregulation of bradykinin, leading to nitric oxide
synthesis,29,30 which may be important in resistance to acute
T cruzi–induced myocarditis.31 Lastly, the thiol group of
captopril is thought to ameliorate encephalomyocarditis virus–induced myocarditis by elimination of oxygen radicals.32
We are currently investigating these 3 possibilities.
Acknowledgments
This work was supported in part by grants from the US Public Health
Service. J.S. Leon was supported by a predoctoral fellowship from
the American Heart Association, Midwest Affiliate. We thank Dr A.
Rademaker for advice on statistical analysis and Dr A. Molteni for
the gift of captopril.
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