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Journal of Virology, January 2001, p. 351-361, Vol. 75, No. 1
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.351-361.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Calpain Inhibition Protects against Virus-Induced
Apoptotic Myocardial Injury
Roberta L.
DeBiasi,1,2,3
Charles L.
Edelstein,4
Barbara
Sherry,5 and
Kenneth L.
Tyler2,3,4,6,*
Departments of Pediatric Infectious
Diseases,1
Neurology,2
Medicine,4 and Microbiology and
Immunology,6 University of Colorado Health
Sciences Center, and Denver Veterans Affairs Medical
Center,3 Denver, Colorado 80262, and
Department of Microbiology, College of Veterinary Medicine,
North Carolina State University, Raleigh, North Carolina
276065
Received 23 May 2000/Accepted 14 September 2000
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ABSTRACT |
Viral myocarditis is an important cause of human morbidity and
mortality for which reliable and effective therapy is lacking. Using
reovirus strain 8B infection of neonatal mice, a well-characterized experimental model of direct virus-induced myocarditis, we now demonstrate that myocardial injury results from apoptosis. Proteases play a critical role as effectors of apoptosis. The activity of the
cysteine protease calpain increases in reovirus-infected myocardiocytes and can be inhibited by the dipeptide alpha-ketoamide calpain inhibitor
Z-Leu-aminobutyric acid-CONH(CH2)3-morpholine
(CX295). Treatment of reovirus-infected neonatal mice with CX295
protects them against reovirus myocarditis as documented by (i) a
dramatic reduction in histopathologic evidence of myocardial injury,
(ii) complete inhibition of apoptotic myocardial cell death as
identified by terminal deoxynucleotidyltransferase-mediated dUTP-biotin
nick end labeling, (iii) a reduction in serum creatine phosphokinase, and (iv) improved weight gain. These findings are the first evidence for the importance of a calpain-associated pathway of apoptotic cell
death in viral disease. Inhibition of apoptotic signaling pathways may
be an effective strategy for the treatment of viral disease in general
and viral myocarditis in particular.
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INTRODUCTION |
The mechanisms by which viruses
produce cytopathic effects in their host cells are not well understood.
Such knowledge is essential to an understanding of viral pathogenesis
and development of novel antiviral therapies. Apoptosis is a mechanism
of active cell death distinct from necrosis, characterized by DNA
fragmentation, cell shrinkage, and membrane blebbing without rupture
(26). Apoptosis plays a critical role in many physiologic
(28, 74), as well as infectious and noninfectious,
pathologic conditions (72). Viruses may either promote or
inhibit apoptosis as a strategy to maximize pathogenicity in their
hosts (40, 54, 67). Several viruses, including adenovirus,
poxviruses, herpesviruses, and human papillomavirus, proliferate and
evade host immune responses by interfering with programmed cell death
(1, 19, 31, 68). Many other viruses, such as human
immunodeficiency virus, human T-cell leukemia virus, influenza virus,
measles virus, rubella virus, poliovirus, human herpesvirus 6, Sindbis
virus, and reoviruses, cause cytopathic effect by induction of
apoptosis in their target cells (11, 14, 21-23, 34, 40, 42, 50,
70).
We have used reovirus-induced apoptosis as an experimental model system
to study the viral and cellular mechanisms involved in apoptotic cell
death (39). Reoviruses are nonenveloped viruses that
contain a genome of segmented, double-stranded RNA. Infection of
cultured fibroblasts and epithelial cells with reoviruses induces apoptosis. Reoviral strains differ in the efficiencies with which they
induce this cellular response, and these differences are determined by
the viral S1 gene (44, 69). Apoptosis also occurs following reovirus infection in vivo and colocalizes with areas of
pathologic injury (38, 39). This finding suggests that apoptosis is an important mechanism of tissue damage in reoviral infection.
Reovirus strain 8B is a reassortant reovirus that efficiently produces
myocarditis in infected neonatal mice (55, 58). Damage has
been shown to be a direct effect of viral infection of myocardiocytes
(60). This damage differs from that of several other
models of viral myocarditis (such as coxsackievirus and murine
cytomegalovirus) in which secondary inflammatory responses, or
lymphocyte recognition of viral or self-antigens on myocardial cells,
may be the predominant cause of cardiac damage (12, 17, 20, 30,
46). SCID mice infected with reovirus 8B develop myocarditis,
and passive transfer of reovirus-specific immune cells is protective,
rather than harmful, to 8B-infected mice (58, 60). This
finding indicates that immune mechanisms contribute to amelioration
rather than induction of reovirus-induced viral injury
(60). However, the mechanism by which direct myocardial injury occurs is not well characterized. Since tissue damage occurs by
apoptosis in other in vivo models of reoviral infection
(38), and apoptosis has been suggested in some models of
viral myocarditis (6, 25), we wished to determine if
reoviral myocarditis occurs as a result of apoptotic cell injury and,
if so, whether manipulation of known signaling pathways preceding
apoptosis is protective.
Protease cascades appear to play critical roles as effectors of
apoptosis, as with the cysteine proteases caspases and calpain (10, 32, 41, 62, 79). Caspases are the most extensively investigated members of this class of protease and have been implicated in a wide variety of apoptotic models. However, the role of calpain in
apoptosis has been recognized recently. Calpain is a calcium-dependent neutral cysteine protease that is ubiquitous in the cytosols of many
cell types (35, 63). Calpains have recently been
implicated in several models of apoptosis, including
dexamethasone-induced thymocyte apoptosis (65), neuronal
cell apoptosis (36), neutrophil apoptosis
(64), ischemia-induced rat liver apoptosis (27, 61), myonuclear apoptosis in limb-girdle dystrophy
(3), and chemical hypoxia-induced apoptosis of rat
myocytes (8). We have recently shown that reovirus-induced
apoptosis in vitro is preceded by increased cellular calpain activity
and is inhibited by two classes of calpain inhibitors
(13).
We now show that reovirus 8B-induced myocarditis occurs by apoptosis.
Calpain activity increases in cardiomyocytes following infection with
reovirus 8B, and calpain inhibition reduces myocardial injury and
morbidity in infected mice. This is evidence that interference with
apoptotic signaling pathways may prove of benefit as a therapeutic strategy in the treatment of viral infection in general and viral myocarditis in particular.
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MATERIALS AND METHODS |
Virus.
Reovirus 8B is an efficiently myocarditic reovirus
that has been previously characterized (58). 8B stocks
were subjected to plaque assay three times and passaged twice in mouse
L cells prior to use.
Mice.
Swiss-Webster (Taconic) mouse litters were housed in
individual filter-topped cages in an American Association for
Laboratory Animal Care-accredited animal facility. All animal
procedures were performed under protocols approved by the appropriate
institutional committees.
Mouse inoculations.
Two-day-old Swiss-Webster (Taconic) mice
were intramuscularly inoculated with 1,000 PFU of 8B reovirus in the
left hind limb (20-µl volume). Mock-infected mice received gel saline
vehicle inoculation (equal volume) (137 mM NaCl, 0.2 mM
CaCl2, 0.8 mM MgCl2, 19 mM
H3BO3, 0.1 mM
Na2B4O7, 0.3% gelatin).
Histologic analysis.
At 7 days postinfection, mice were
sacrificed and hearts were immediately immersed in 10% buffered
formalin solution. After being mounted as transverse sections, hearts
were embedded in paraffin and sectioned to 6 µm in thickness. For
quantification of degree of myocardial injury, hematoxylin- and
eosin-stained midcardiac sections (at least six per heart) were
examined at a ×125 magnification by light microscopy and scored
blindly. Scoring was performed using a previously validated system
(58), with scores ranging from 0 to 4 (0, no lesions; 1, one or a few small lesions; 2, many small or a few large lesions; 3, multiple small and large lesions; and 4, massive lesions). Twenty-three
to 24 mice were scored from each group.
DNA fragmentation.
The presence of internucleosomal DNA
cleavage in myocardial tissue was investigated by phenol-chloroform
extraction of DNAs from 8B-infected and mock-infected hearts and
precipitation in 95% ethyl alcohol. The DNA was then end labeled with
5 µCi of [32P]dGTP using 10 U of terminal transferase
(M187; Promega Corporation), resolved by electrophoresis on a 2%
agarose gel, fixed in 5% acetic acid-5% methanol, dried, and scanned
on a Instant Imager (Packard Instrument Company).
TUNEL.
Evaluation of fragmented DNA was performed by
terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick
end labeling (TUNEL), as previously described (38).
Paraffin-embedded cardiac midsections were prepared by removing
paraffin with xylene and then rehydrating them in 100, 95, and then
70% ethanol solutions. After digestion in proteinase K solution
(Boehringer Mannheim) for 30 min at 37°C, slides were pretreated in
0.3% H2O2 in phosphate-buffered saline for 15 min at room temperature and then washed. The TdT labeling reaction was
carried out under coverslips in a humidified chamber for 1 h at
37°C with TdT and digoxigenin 11-dUTP. (Boehringer Mannheim). The
reaction was stopped with SSC (1× SSC is 0.15 M NaCl plus 0.015 M
sodium citrate) buffer. After being blocked in 2% bovine serum albumin
for 10 min, sections were probed with Vectastain ABC (avidin DH and
biotinylated enzyme; Vector Laboratories) for 1 h at room
temperature, and then visualized with a diaminobenzadine peroxidase
substrate kit (Vector Laboratories). Negative and positive controls
were used with all reactions.
Viral-antigen stain.
Cardiac midsections were prepared as
noted above. Following the hydrogen peroxide incubation, slides were
blocked in 2% normal goat serum for 30 min at room temperature.
Sections were then incubated in rabbit polyclonal anti-reovirus type 3 Dearing antiserum as the primary antibody (gift of Terence Dermody,
Vanderbilt University) at a dilution of 1:1,000 for 1 h at 37°C.
Biotinylated goat anti-rabbit antibody was used as the secondary
antiserum (1:200 dilution in 2% normal goat serum) for 30 min at
37°C. Sections were probed and visualized as noted above.
Calpain activity in myocytes.
The determination of the
presence of calpain-specific spectrin (fodrin) breakdown products (150- and 145-kDa doublet) by immunoblotting was used as an assay of calpain
activity (36). Mouse primary cardiac myocyte cultures were
prepared as previously described (5). Cells were plated at
1.6 × 106 cells/well in 24-well plates and incubated
for 48 h. Cells were then infected with reovirus strain 8B
(multiplicity of infection [MOI], 20, in Dulbecco modified Eagle
medium [DMEM]) or mock infected (DMEM) and then incubated at 37°C.
Mock-infected cells were harvested at 48 h. 8B-infected cells were
harvested at 24, 48, and 72 h postinfection. Cell lysates were
prepared by sonication in lysis buffer (15 mM Tris [pH 7.4], 10 mM
EDTA, 0.1% NP-40, 20% glycerol, 50 mM 
-mercaptoethanol, 50 µg of
pepstatin per ml, 100 µg of leupeptin per ml, 1 mM
phenylmethylsulfonyl fluoride), and the cytoplasmic fractions were run
on a 7.5% polyacrylamide gel. Protein loading for these gels (25 µg/well) was normalized by protein concentration analysis of cell
lysates. Following transfer (15 V overnight), the nitrocellulose
membrane was blocked in 5% nonfat dried milk-Tris normal saline for
2 h, probed with anti-fodrin mouse monoclonal antibody (ICN) at a
dilution of 1:1,000 for 1.5 h, and then washed. Membranes were
then incubated in anti-mouse immunoglobulin G horseradish
peroxidase-linked whole antibody (Amersham) at a dilution of 1:1,000,
as the secondary antibody. After the membranes were washed, ECL Plus
(Amersham) was used for detection.
In additional experiments, primary cardiac myocytes were infected with
8B reovirus (using the method described above) with and without
pretreatment in CX295 (100 µM). Cell lysates were prepared and
analyzed for calpain activity by immunoblotting as described above.
Specificity of the calpain inhibitor CX295.
Z-Leu-aminobutyric
acid- CONH(CH2)3-morpholine (CX295) is a dipeptide
alpha-ketoamide compound which inhibits calpain at the active site
(kindly provided by Gary Rogers at Cortex Pharmaceuticals, Inc.). To
determine the efficacy of CX295 as a calpain inhibitor, 10 µg of
purified µ-calpain (porcine RBC; Calbiochem) was added to the
preferred fluorogenic calpain substrate
sucrose-Leu-Tyr-amino-methyl-coumarin (SLY-AMC) in the presence and
absence of CX295 (100 µM), as well as in the presence of the
pan-caspase inhibitor Z-D-DCB (100 µM). The
calpain assay was performed as previously described (16). Proteolytic hydrolysis of the substrate by purified calpain liberates the highly fluorescent AMC moiety. Fluorescence at a 380-nm excitation and 460-nm emission was quantified with a Hitachi F2000
spectrophotometer. An AMC standard curve was determined for each
experiment. Calpain activity was expressed in picomoles of AMC released
per minute of incubation time per microgram of purified calpain.
To determine the specificity of CX295 as a calpain inhibitor, 10 ng of
purified caspase 3 (Upstate) was added to the preferred
fluorogenic
caspase-3 substrate DEVD-AMC in the presence and absence
of the
pan-caspase inhibitor
Z-
D-DCB (100 µM), as
well as CX295
(100 µM). In addition, 57 ng of purified caspase-1
(provided by
Nancy Thornberry, Merck) was added to the preferred
fluorogenic
caspase-1 substrate YVAD-AMC in the presence and absence of
Z-
D-DCB,
as well as CX295. The caspase activity
assay was performed as
previously described (
16). Caspase
activity was expressed as
picomoles of AMC released per minute of
incubation time per nanogram
of purified caspase. Experiments were all
performed in
triplicate.
Calpain inhibition in vivo.
For calpain inhibition
experiments, animals received daily intraperitoneal injections of
either active CX295 (70 mg/kg of body weight in a 50-µl volume) or
its inactive saline diluent. The first dose was given 30 min prior to
infection with 8B virus. A total of six doses were given, at 24-h
intervals. Mice were sacrificed at 7 days postinfection.
Viral titer determination.
Injected hind limbs and whole
hearts were placed in 1 ml of gel saline and immediately frozen at
70°C. After three freeze (70°C)-thaw (37°C) cycles, the
tissues were sonicated approximately 15 to 30 s by using a
microtip probe (Heat Systems model XL2020) until a homogenous solution
was obtained. The virus suspensions were serially diluted in 10-fold
steps in gel saline and placed in duplicate on L-cell monolayers for
plaque assay, as previously described (13). Virus titers
were expressed as log10 PFU per milliliter.
Serum CPK.
Following decapitation of mice, whole blood was
collected from individual mice into plasma separator tubes with lithium
heparin to prevent coagulation (Microtainer; Becton Dickinson). Samples were collected from 8B-infected mice treated with CX295 (n = 20), 8B-infected mice treated with the inactive diluent
(n = 20), and uninfected age-matched controls
(n = 9). Serum creatine phosphokinase (CPK)
measurements were performed by the University of Colorado Health
Sciences Center Clinical Laboratory and were expressed as units per liter.
Growth.
Mice were infected with 10 PFU of 8B reovirus.
Infected drug-treated (n = 15) and infected control
(n = 15) mice were weighed daily on days 0 to 14 postinfection. Additional experiments using a higher dose of virus
(1,000 PFU) were also completed, with daily weighing on days 0 to 7 postinfection. In these experiments, weights were also compared to
those of normal age-matched uninfected mice.
Statistics.
The results of all experiments are reported as
means ± standard errors of the means. Means were compared using
parametric two-tailed t tests (Graph Pad; Prism), and
differences were considered significant if P values were
<0.05.
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RESULTS |
(i) Reovirus 8B induces myocardial injury by apoptosis.
Mice
were inoculated intramuscularly into the hind limb with either 1,000 PFU of strain 8B reovirus or gel saline (mock infection). At 7 days
postinfection, mice were sacrificed and transverse cardiac sections
were prepared for histologic evaluation. In the 8B-infected hearts,
there was marked myocardial disruption and diffuse edema (Fig.
1B). Numerous pyknotic nuclei and
apoptotic bodies were present (Fig. 1B; see also Fig. 5E). Despite the
degree of myocardial injury, only rare mononuclear cells (predominantly
macrophages) and neutrophils were present. Myocardial disruption and
edema were not seen in mock-infected hearts (Fig. 1A). Extensive areas of apoptotic TUNEL-positive nuclei were noted in the 8B-infected hearts
(Fig. 1D), which correlated with the areas of histologic abnormality
described above. Mock-infected hearts were TUNEL negative (Fig. 1C).
Large regions of reovirus antigen-positive tissue were noted in the
8B-infected hearts (Fig. 1F) and occurred in the same distribution as
the areas of histologic injury and TUNEL-positive cells.
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FIG. 1.
Consecutive cardiac midsections from mock-infected (A,
C, and E) and reovirus 8B-infected (B, D, and F) neonatal mice 7 days
after left hind limb inoculation with 1,000 PFU of strain 8B reovirus
or mock inoculation. Hematoxylin- and eosin-stained tissue reveals
marked disruption of myocardial architecture in the 8B-infected heart
(B) compared to that in the mock-infected heart (A). Despite the degree
of injury, there is minimal inflammatory cell infiltrate. The degree of
cellularity seen in both mock-infected and infected hearts is normal
for neonatal mice. In situ detection of DNA nick ends by TUNEL revealed
positively staining nuclei in the same region of an injured 8B-infected
heart (D), which are absent in a mock-infected animal (C).
Immunohistochemistry with anti-type 3 Dearing reovirus antibody reveals
the presence of viral antigen in the areas of myocardial injury in the
8B-infected mouse (F), absent in the mock-infected animal (E). Original
magnification, ×25.
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In order to provide further confirmation that the morphological changes
seen in virus-infected hearts were indeed due to apoptosis,
DNAs were
extracted from 8B-infected and mock-infected hearts,
end labeled, and
analyzed by agarose gel electrophoreses. DNAs
from infected hearts, but
not mock-infected controls, showed fragmentation
into
oligonucleosomal-length ladders, characteristic of apoptosis
(Fig.
2). Taken together, these findings
indicate that reovirus-induced
myocardial injury is due to apoptosis.
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FIG. 2.
DNA laddering in 8B-infected-neonatal-mouse hearts. DNA
was extracted from reovirus 8B-infected and mock-infected hearts and
detected by end-labeling analysis. DNA from the heart of a
representative 8B-infected mouse (lane 2) is fragmented into
oligonucleosomal-length pieces. These fragments result from
internucleosomal DNA cleavage, a hallmark of apoptosis. (Arrowheads
indicate DNA fragments, ranging in length from 200 to 1,000 bp).
Fragmentation is absent in the mock-infected animal (lane 1).
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(ii) Calpain is activated in 8B-infected murine cardiac
myocytes.
We have previously shown that reovirus infection in L929
cells results in increased calpain activity, which precedes apoptosis (13). We wished to determine whether calpain activity was
also increased in myocardial cells following reovirus infection.
Proteolysis of spectrin (fodrin), a preferred calpain substrate, was
examined as an indication of calpain activation. Intact spectrin (280 kDa) is degraded by calpain, resulting in a characteristic spectrin breakdown product doublet seen at 150 and 145 kDa. Mouse primary cardiac myocyte cultures were infected with reovirus strain 8B (MOI,
20) or mock infected. Lysates were prepared after harvesting of cells
at 24, 48, and 72 h postinfection. Western blot analysis of these
cytoplasmic fractions revealed increased calpain activity following 8B
infection compared to that of mock-infected cytoplasmic fractions. This
increase was first detectable at 24 h, reached maximal intensity
by 48 h, and was still present at 72 h postinfection (Fig.
3A). Densitometric analysis of
calpain-specific breakdown products revealed a peak fourfold increase
in calpain activity following virus infection (Fig. 3B). Caspase
degradation of spectrin results in breakdown products at 120 kDa, which
were not observed in this experiment. These data demonstrate that an
increase in calpain activity occurs following infection of
myocardiocytes with reovirus strain 8B, in a time course that parallels
the onset of apoptosis.
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FIG. 3.
Calpain activity in a reovirus-infected primary cardiac
myocyte culture. (A) Activity was measured by monitoring proteolysis of
spectrin (280 kDa), a preferred calpain substrate, to its
calpain-specific breakdown products (150- to 145-kDa doublet) by
immunoblot assay. Lysates of 8B-infected cardiomyocytes (MOI, 20) were
prepared at 24, 48, and 72 h postinfection and compared to lysates
of mock-infected cells at 48 h postinfection (experiments with all
conditions were performed in duplicate). Compared to that of
mock-infected mice, 8B-infected mice had increased calpain activity
(150- to 145-kDa doublet) beginning at 24 h postinfection, which
was markedly increased at 48 h postinfection and still present,
but decreasing, at 72 h postinfection. No change was detectable in
the intact (280-kDa) spectrin band following infection. (B)
Densitometric analysis of calpain-specific spectrin breakdown products
(150- to 145-kDa doublet) reveals a peak fourfold increase in calpain
activity following infection with 8B reovirus.
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(iii) CX295 is an effective calpain inhibitor and does not
appreciably inhibit caspases.
Based on our findings that (i)
8B-induced myocarditis is due to apoptosis, (ii) calpain is activated
in 8B-infected myocardiocytes, and (iii) inhibition of calpain
activation inhibits reovirus-induced apoptosis in vitro, we wished to
determine if calpain inhibition could prevent 8B-induced myocardial
injury in vivo. CX295 is a dipeptide alpha-ketoamide compound that
inhibits calpain at the active site and is nontoxic and effective in
vivo (4, 48). To confirm the specificity of the compound,
the activity of purified calpain was monitored in the presence and
absence of CX295. The inactive diluent of CX295 and the pan-caspase
inhibitor Z-D-DCB were used as controls.
Purified calpain activity (measured as cleavage of SLY-AMC) was
markedly reduced by CX295 (Fig. 4A). Calpain activity was decreased from 1,255 ± 22 to 206 ± 4 pmol/min/µg in the presence of CX295 (P < 0.0001).
Neither the diluent nor Z-D-DCB controls had
appreciable effect on calpain activity.
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FIG. 4.
Efficacy and specificity of the calpain inhibitor CX295.
The effects of the calpain inhibitor CX295 on purified calpain (A),
caspase 3 (B), and caspase 1 (C) activity were evaluated using a
fluorogenic substrate assay, in which SLY-AMC, DEVD-AMC, and YVAD-AMC,
respectively, served as substrates. Activities are expressed as
purified calpain or caspase activity per minute, per micromole or
picomole of substrate. One hundred micro-molar CX295 significantly
inhibited calpain activity (the asterisk indicates a P of
<0.0001) (A) but had minimal effect on caspase 3 or caspase 1 activity. Z-D-DCB, a pan-caspase inhibitor, was
used as a control. Z-D-DCB had no effect on
calpain activity but nearly completely inhibited caspase 3 and 1 activities (B and C). CX295 also effectively inhibited calpain activity
in 8B-infected cardiomyocytes in vitro (D). Calpain activity (measured
by densitometric analysis of the 150- to 145-kDa calpain-specific
fodrin degradation product) increased by 2.4-fold in 8B-infected
cardiomyocytes compared to that in mock-infected cardiomyocytes.
Calpain activity was significantly reduced in CX295-treated,
8B-infected cells compared to that in infected, untreated cells
(P = 0.04). (The 150- to 145-kDa doublet appears as a
single large band due to the gel conditions.)
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To further confirm that CX295 is a specific calpain inhibitor and does
not inhibit caspases, the activities of purified caspase
1 and caspase
3 were monitored in the presence and absence of
CX295, as well as in
the presence of the known pan-caspase inhibitor
Z-
D-DCB (positive control). CX295 had a minimal
inhibitory effect
on either caspase 1 or caspase 3 activity (measured
as cleavage
of YVAD-AMC and DEVD-AMC, respectively), whereas
Z-
D-DCB inhibited
both caspases nearly
completely (Fig.
4B and C). Purified caspase
1 activity decreased from
6,829 ± 39 to 6,496 ± 36 pmol/min/ng
in the presence of
CX295, compared to 0 pmol/min/ng in the presence
of
Z-
D-DCB. Purified caspase 3 activity decreased
from 1,752 ±
6 to 1,533 ± 9 pmol/min/ng in the presence of
CX95, compared to
118 ± 4 pmol/min/ng in the presence of
Z-
D-DCB.
CX295 also effectively inhibited calpain activity in primary
cardiomyocyte culture. Calpain activity was quantified by
immunoblotting
in reovirus-infected myocytes, in the presence and
absence of
CX295. Calpain activity (measured by densitometric analysis
of
the 150- and 145-kDa calpain-specific fodrin breakdown product)
increased by 2.4-fold in 8B-infected cardiomyocytes compared to
that in
mock-infected cardiomyocytes. Calpain activity was significantly
reduced in CX295-treated, 8B-infected cells compared to that in
infected, untreated cells (
P = 0.04) (Fig.
4D and
E).
These experiments confirm that CX295 is both an effective and a
specific calpain
inhibitor.
(iv) Calpain inhibitor CX295 inhibits 8B-induced myocardial
injury.
Two-day-old Swiss-Webster mice were infected with 1,000 PFU of 8B virus and received six daily intraperitoneal injections of
either active CX295 (70 mg/kg) or its inactive saline diluent as a
control (see Materials and Methods). Mice were sacrificed on day 7 postinfection, and myocardial sections were prepared.
Transverse cardiac sections from 8B-infected mice treated with CX295 or
its inactive diluent (control) were stained with hematoxylin
and eosin
and viewed by light microscopy (Fig.
5A to F). Cardiac
tissue from
control mice (Fig.
5A)
showed extensive focal areas
of myocardial damage, which were absent in
the CX295-treated animals,
despite identical viral infection (Fig.
5B).
Marked disruption
of the normal myocardial architecture was evident in
hearts of
control mice (Fig.
5C), compared to that of drug-treated
animals
(Fig.
5D). Nuclei with apoptotic morphology were easily seen
within
these areas in the control mice (Fig.
5E), including in cells
with condensed and pyknotic nuclei, as well as apoptotic bodies.
These
characteristics were absent in the drug-treated animals
(Fig.
5F).
Staining by TUNEL of comparable sections from drug-treated
and
diluent-treated (control) infected mice was examined. Nuclei
that
stained positive by TUNEL were virtually absent in the drug-treated
mice (Fig.
5H), unlike with control infected animals (Fig.
5G).
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FIG. 5.
Cardiac midsections from reovirus 8B-infected
neonatal mice treated with the calpain inhibitor CX295 (B, D, F, and H)
compared to those from inactive diluent control mice (A, C, E, and G) 7 days following intramuscular inoculation with 1,000 PFU of reovirus 8B.
Hematoxylin- and eosin-stained sections at an original magnification of
×25 reveal extensive focal areas of myocardial injury (arrows) in the
control animal (A), which is absent in the CX295-treated animal (B),
despite identical viral infections. Views at an original magnification
of ×50 demonstrate minimal inflammatory cell infiltrate in the
affected area (C), but myocardial architecture is dramatically
disrupted, compared to that of a CX295-treated mouse (D). At an
original magnification of ×100, nuclei with apoptotic morphology are
easily seen in the control animal (E), as are cells with
condensed and pyknotic nuclei (long arrow) as well as apoptotic
bodies (shorter arrows). These characteristics are absent in the
drug-treated mouse (F). TUNEL analysis of the control animal reveals
extensive areas of positively staining cells in the same regions of
injury (G) but no TUNEL-positive areas in the drug-treated mouse (H).
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For quantification of the degree of myocardial injury, hematoxylin- and
eosin-stained midcardiac sections (at least six sections
per heart)
were scored using a previously validated scoring system
(
58). Twenty infected, CX295-treated animals and 23 control
(infected, diluent-treated) mice were evaluated. There was a
highly
significant reduction in the myocardial injury scores of animals
treated with CX295. The mean score for control animals was 3.0
± 0.1 (range, 2 to 4), compared to 0.6 ± 0.1 (range 0 to 1.5)
for
CX295-treated animals (
P < 0.0001) (Fig.
6).
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FIG. 6.
Reduction in myocardial injury score. Myocardial injury
of 8B-infected animals was quantified by blindly scoring hematoxylin-
and eosin-stained midcardiac sections of drug-treated and control
(inactive-diluent-treated) animals upon light microscopy. At least six
sections per heart were scored from 20 to 23 animals in each group. A
highly significant reduction in myocardial injury score was noted for
drug-treated animals. The mean lesion score was 3.0 ± 0.1 (range
2 to 4) for controls, compared to 0.6 ± 0.1 (range 0 to 1.5) for
CX295-treated animals. *, P < 0.0001. See Materials
and Methods for explanations of mean lesion scores.
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CPK is an intracellular enzyme present in cardiac and skeletal muscle
that is released upon tissue injury. It can be measured
in the serum
and used as a quantitative marker of skeletal and
cardiac muscle damage
(
2). Blood was collected from infected
mice treated with
CX295 and inactive diluent-treated controls
at 7 days postinfection, as
well as uninfected age-matched mice.
Serum CPK levels were
significantly elevated in 8B-inoculated
mice compared to those in
uninfected mice, indicative of 8B-induced
muscle injury. There was a
statistically significant reduction
in serum CPK level toward a normal
level in CX295-treated mice
compared to the level in control mice (Fig.
7). Uninfected age-matched
mice had a
mean CPK level of 4,521 ± 431 U/liter. 8B-infected
mice had a
mean CPK level of 5,658 ± 359 U/liter, representing
an increase
of 1,137 U/liter above normal. Treatment of infected
animals with CX295
reduced the mean CPK value to 4,634 ± 350 U/liter,
not
significantly elevated compared to normal levels but significantly
reduced compared to levels in infected, nontreated animals
(
P < 0.05).
View larger version (22K):
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|
FIG. 7.
Reduction in serum CPK. CPK was measured as a marker of
myocardial damage in 8B-infected animals treated with CX295, and levels
were compared to those for control (inactive-diluent-treated) infected
animals, as well as age-matched uninfected controls. There was a
significant reduction in serum CPK toward normal levels in
CX295-treated infected mice compared to levels in infected control
animals (P < 0.05).
|
|
To determine if reductions in myocardial injury were also associated
with reduction in viral titer at primary (hind limb)
and secondary
(heart) sites of replication, the titers of virus
in tissues were
determined by plaque assay of tissue homogenates.
There was a
0.5-log
10-PFU/ml reduction in viral titers in the
hind
limbs of CX295-treated animals compared to those for controls
(7.2 ± 0.1 to 6.7 ± 0.2 log
10 PFU/ml;
P = 0.003). Hearts of CX295-treated
animals had a
0.7-log
10-PFU/ml reduction in viral titer (6.1 ±
0.2 to 5.4 ± 0.2 log
10 PFU/ml;
P < 0.01)
(Fig.
8). Although these
decrements were
statistically significant, they were modest in
degree, and substantial
viral replication occurred in both the
drug-treated and the control
animals (1,000- to 10,000-fold).
View larger version (33K):
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[in a new window]
|
FIG. 8.
Tissue-specific viral titers. Titers were measured by
plaque assay at 7 days postinfection from homogenates of limbs (site of
primary replication) and hearts (site of secondary replication) of
8B-infected mice. A slight reduction in peak viral titers was seen in
the CX295-treated group. Both CX295-treated and control animals showed
a >3-log10-unit increase in virus over the input inoculum
(103 PFU/mouse).
|
|
We wished to determine whether the reduction in myocardial damage
caused by CX295 treatment reduced morbidity in mice. We
therefore
measured growth (weight gain) in infected, drug-treated
mice and
compared to that in infected, untreated controls. CX295-treated
mice
had improved growth compared to that of control mice (4.6
± 0.4 versus 3.6 ± 0.1 g at 7 days postinfection;
P = 0.008),
and growth was not significantly different from that of
uninfected
age-matched animals (4.6 ± 0.4 versus 4.8 ± 0.1 g;
P = 0.6, not
significant) (Fig.
9).
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 9.
Growth of infected mice. Growth, as measured by weight
gain, was assessed in 8B-infected mice treated with CX295 and inactive
diluent (control). Improved growth was noted for infected,
CX295-treated animals at 7 days postinfection compared to the growth of
infected controls (*, P = 0.008). Weights of
infected, treated animals were not significantly different from those
of uninfected, age-matched controls.
|
|
| |
DISCUSSION |
Viral myocarditis remains a serious disease without reliable or
effective treatment. The events following viral attachment and
replication in myocardial tissue that lead to myocarditis are not
clearly understood. A variety of mechanisms from various models have
been suggested, including direct viral injury and persistence (9,
24), autoimmune phenomena (17, 45, 51), cytokine
fluxes (18, 33, 52, 59), inflammation (29, 53), and apoptosis (12, 77, 78). A clearer
understanding of pathogenic mechanisms is crucial for the development
of effective therapeutic strategies, since currently employed antiviral
agents have not made a significant impact on outcomes from this
clinical syndrome. Reoviral myocarditis is an ideal model with which to study these events, since myocardial injury is a direct effect of virus
infection and does not involve immune-mediated effects.
Reovirus 8B induces myocarditis by apoptosis.
Reovirus 8B
induces myocarditis in mice by direct viral injury to myocytes, and we
now show that this occurs by induction of apoptosis. This conclusion is
supported by the presence of distinctive morphologic criteria upon
microscopic examination of infected heart tissues; TUNEL-positive
nuclei were found exclusively in regions of viral infection and
myocardial injury. The presence of apoptosis was confirmed by the
presence of the characteristic intranucleosomal cleavage pattern of
extracted DNA. It has been shown previously that multiple viral genes
(M1, L1 and L2, and S1) encoding core and attachment proteins are
determinants of reovirus-induced acute myocarditis (56).
Interactions between these proteins determine myocarditic potential.
Several of these genes have been associated with reovirus RNA synthesis
and reovirus induction of and sensitivity to beta interferon in cardiac
myocyte cultures, which are determinants of reovirus myocarditic
potential. In addition, the S1 gene, which codes for the viral
attachment protein 
1, is the primary determinant of apoptotic
potential among strains of reovirus (69). It is likely,
therefore, that the extent of reovirus-induced myocardial injury is
determined by a combination of host responses, encompassing both the
interferon and the apoptosis pathways. Indeed, just as inhibition of
the interferon pathway was sufficient to enhance reovirus-induced myocarditis (59), we show here that inhibition of the
apoptotic pathway is sufficient to abrogate reovirus-induced
myocarditis. Thus, apoptosis is an integral component of
reovirus-induced myocardial injury.
Calpain activity is increased in reovirus-infected myocardiocytes,
and calpain inhibition is protective against reovirus-induced
myocarditis.
Calpain is a calcium-activated cysteine protease that
has proven importance with regard to the initiation of apoptosis in the
reoviral model, as well as several other unrelated models of apoptosis
(see the introduction). We first demonstrated that calpain activity is
increased in cardiac myocytes in vitro following 8B infection, in a
time course paralleling induction of apoptosis. We then demonstrated
that calpain inhibition resulted in a reduction of calpain activity in
infected cells and dramatic reductions in reovirus-induced injury, as
well as apoptosis. Clinically significant reduction in myocardial
injury was documented by reduced serum CPK levels and improved growth
in treated mice.
It is likely that CX295 acted primarily by interfering with crucial
signal transduction cascades involving calpain, required
for induction
of apoptotic cell death. Additionally, it is possible
that CX295
provided some portion of its effect by inhibiting viral
growth at
either the primary (hind limb) or secondary (heart)
site of
replication, since viral titers were slightly lower in
drug-treated
animals than in controls. Slight reductions in viral
titer do not seem
a likely explanation for the majority of drug
effect, since in prior
experiments involving reovirus-induced
myocarditis, yields of virus at
early and late times postinfection
did not correlate with the degree of
myocardial injury (
57).
In addition, efficiently
myocarditic and poorly myocarditic reovirus
strains replicate to
similar titers in the heart; thus, differences
in myocarditic potential
do not simply reflect viral growth in
the heart (
56).
One must be cautious in attributing a role for calpain in disease
pathogenesis based solely on data derived from calpain inhibition.
Currently available calpain inhibitors suitable for in vivo use
have
weak, but measurable, inhibitory activities against other
cysteine
proteases. However, the inhibitor employed in our experiments,
CX295,
is 500- to 900-fold more active against calpains than cathepsins
(the
Ki for calpain is 0.027
0.042 µM,
versus a
Ki for cathepsin
B of 24 µM) and
failed to inhibit caspase activity in vitro, as
described in this
paper. We believe that inhibition of calpain,
rather than of other
cysteine proteases, is the essential element
of CX295's protective
effect against 8B-induced myocardial apoptosis
and
injury.
It is not clear what constitutes the upstream and downstream components
of a signaling cascade within which calpain might
fit, either during
reovirus infection or in other systems where
calpain is involved. The
mechanisms by which reovirus triggers
increased cellular calpain
activity are not known but may include
initiation of calcium fluxes
following viral attachment, as demonstrated
with rotavirus, a closely
related virus (
15); upregulation of
growth factors which
facilitate calpain activation (
37,
66);
or upregulation of
endogenous calpain activator proteins which
have been characterized for
several cell types (
49). Calpain
may play a physiologic
role in the regulation of a variety of
cellular transcription factors
and cell cycle-regulating factors
implicated in apoptosis, including
Jun, Fos, p53, cyclin D, and
NF-
B (
3,
7,
73). We have
recently shown that activation
of NF-
B is required for
reovirus-induced apoptosis (J. L. Connolly,
S. E. Rodgers, B. Pike, P. Clarke, K. L. Tyler, and T. S. Dermody,
Abstr. 17th
Ann. Meet. Am. Soc. Virology, abstr. 17-2, 1998),
suggesting the
possibility that calpain inhibition acts to modulate
NF-
B-induced
signal transduction. Calpains may also modulate
cell death by cleaving
Bax, a proapoptotic protein located in
the cytosol (
75).
In addition, the caspase and calpain proteolytic
cascades may interact.
Caspases may play a role in the regulation
of calpain by cleavage of
calpastatin, the endogenous inhibitor
of calpain (
43,
71,
76). Reflexively, calpain may be involved
in the proteolytic
activation of some caspases (
47).
Potential therapeutic efficacy of calpain inhibitors.
In
conclusion, our data suggest that reovirus-induced myocarditis occurs
by direct viral induction of apoptotic cell death and that injury can
be markedly reduced with the use of a calpain inhibitor. To our
knowledge, this is the first successful demonstration of the use of
calpain inhibition in vivo to ameliorate myocarditis in particular and
virus-induced disease in general. However, future experiments are
needed to determine whether calpain inhibition remains effective when
it is administered after the onset of viral infection. Our results
demonstrate the utility of apoptosis inhibition as a strategy for
protection against viral infection.
| |
ACKNOWLEDGMENTS |
We thank Gary Rogers of Cortex Pharmaceutical for providing the
calpain inhibitor CX295 and its inactive diluent. The University of
Colorado Cancer Center provided core tissue culture and medium facilities.
This work was supported by Public Health Service grant 1RO1AG14071 from
the National Institute of Aging, Merit and REAP grants from the
Department of Veterans Affairs, a U.S. Army Medical Research and
Material Command grant (USAMRMC 98293015) (K.L.T.), a Young Investigator Award from the National Kidney Foundation (C.L.E.), Public
Health Service grant 1RO1HL57161, and North Carolina State University
College of Veterinary Medicine grant 204743 (B.S.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Neurology (B-182), University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262. Phone: (303) 393-2874. Fax: (303) 393-4686. E-mail: Ken.Tyler{at}UCHSC.edu.
| |
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Journal of Virology, January 2001, p. 351-361, Vol. 75, No. 1
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.351-361.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
