© 2000 American Heart Association, Inc.
Molecular Medicine |
Host Gene Regulation During Coxsackievirus B3 Infection in Mice
Assessment by Microarrays
From the Cardiovascular Research Laboratory (L.A.T., C.M.C., D.Y., K.S., D.W., B.M.M.), Department of Pathology and Laboratory Medicine, University of British Columbia, St. Paul’s Hospital, Vancouver, Canada, and SCIOS, Inc (G.S., L.W.S.), Sunnyvale, Calif.
Correspondence to Bruce McManus, MD, PhD, Department of Pathology and Laboratory Medicine, University of British Columbia, St. Paul’s Hospital, 1081 Burrard St, Vancouver, BC, Canada V6Z 1Y6. E-mail mcmanus{at}interchange.ubc.ca
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Abstract—Host genetic responses that characterize enteroviral myocarditis have not yet been determined. The injurious and inflammatory process in heart muscle may reflect host responses of benefit to the virus and ultimately result in congestive heart failure and dilated cardiomyopathy. On the other hand, host responses within the myocardium may secure the host against acute or protracted damage. To investigate the nature of modified gene expression in comparison with normal tissue, mRNA species were assessed in myocardium using cDNA microarray technology at days 3, 9, and 30 after infection. Of 7000 clones initially screened, 169 known genes had a level of expression significantly different at 1 or more postinfection time points as compared with baseline. The known regulated genes were sorted according to their functional groups and normalized expression patterns and, subsequently, interpreted in the context of viremic, inflammatory, and healing phases of the myocarditic process.
Key Words: coxsackieviruses • gene regulation • microarrays • myocardium
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Nearly 50% of North American clinical myocarditis cases are attributable to Picornaviradae infections, with the coxsackievirus B (CVB) serogroup making up the most significant proportion of such infections.1 Coxsackieviruses generally have a high attack rate with a relatively low myocarditic potential, and a relatively small percentage of the human population develop clinically visible viral myocarditis in a lifetime.2 The susceptible human population appears to be nonrandom, with genotypic factors related to age and sex, and environmental factors such as nutrition and pregnancy, modulating host susceptibility to viral myocarditis. Mouse models of CVB3-mediated myocarditis mimic the human disease process. Indeed, disease progression and pathology closely resemble the human disease,3 and both age and genetic background affect susceptibility.4 5 6 As such, CVB3-mediated murine myocarditis is a valuable model for studying the pathogenesis of virus-induced human myocarditis and has allowed partial dissection of the disease progression of myocarditis, which is otherwise impossible in humans.
Three distinct phases of the infectious and injurious process are appreciated.7 The first phase, encompassing peak viremia, occurs 2 to 4 days after infection wherein direct virus-induced tissue injury and death of infected cardiac myocytes are profound.7 8 Impairment of left ventricular function in physiologically loaded working mouse hearts with histopathologically graded moderate injury after CVB3 infection reinforces the important pathophysiological role of direct viral damage of myocardium. This myocardial injury becomes progressively more noticeable by days 3 and 4 and is significantly advanced by day 5, after which direct injury is complicated by an inflammatory infiltrate, thus marking the beginning of the second phase. This inflammatory infiltrate includes both innate and specific immune participants largely localized to regions of dead, dying, and residually infected myocytes9 and serves to clear both infectious virus particles and dying cells. This phase typically lasts through day 14 after infection and is followed by a healing phase characterized by steady tissue reparation and ventricular remodeling wherein dead myocytes are removed and many remaining myocytes change their structural, ionic, metabolic, and contractile properties.10 In addition, nonmyocytic cells, fibroblasts for example, often increase in number in the cardiac parenchyma during the healing phase, participating in the secretion of cytokines, growth factors, and extracellular matrix (ECM).
The host genetic elements responsible for the changes observed in the above 3 phases of CVB3-mediated myocarditis have not yet been determined. Although previous investigations by our laboratory11 and by others have yielded clues to gene expression involved in the pathology of myocarditis at certain time points in murine models, including those of virus and host, the majority of host genes involved in this complex process remain elusive. Traditional molecular approaches continue to add to our understanding of the pathogenesis and progression of viral myocarditis one gene at a time; however, new technologies in molecular biology, namely cDNA microarrays, have revolutionized the power of unbiased expression searches and afford the opportunity to profile the expression of thousands of genes in a single experiment.12
Thus, to rapidly expand the portrait of host gene expression involved
in the pathogenesis of viral myocarditis and particularly to examine
gene expression during all phases of viral myocarditis, we used cDNA
microarrays to evaluate the relative abundance of myocardial mRNA
species in CVB3-infected male adolescent A/J mice at 3, 9, and 30 days
after infection as compared with sham-infected myocardium.
From an initial screen of 
7000 clones, 614 sequences including 169
known genes were identified as having either a significant (1.8-fold)
decrease or increase in expression at 1 or more postinfection time
points. The 169 known genes were sorted according to their functional
groups including those involved in cell division, structure/motility,
signaling, host defense, gene/protein expression,
metabolism, mitochondrial sequences, and presently
unknown function. These genes were then interpreted in light of known
contributors to the myocarditic process.
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Materials and Methods |
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Animals and Virus
A/J (H-2a) mice (The Jackson Laboratories) were 5 weeks old at the onset of the experiment. Myocarditic CVB3 was kindly provided by Dr C.J. Gauntt (University of Texas, San Antonio, Tex) and stored at –80°C. Virus was propagated in HeLa cells (American Type Culture Collection). Stock virus was titered before the onset of experiments using the plaque assay method. Mice were infected with 1x105 pfu of CVB3 (n=15 per time point) or PBS sham (n=15 per time point) and euthanized by CO2 narcosis on days 3, 9, and 30 after infection. Animals that died naturally after infection (
25%) were
not included in experimental analysis (because of degraded
RNA).
Tissue Histopathology
Heart tissue was fixed in D-PBS–buffered 4%
paraformaldehyde overnight, dehydrated in graded
alcohols, cleared in xylene, embedded in paraffin, and sectioned
(4 µm) for in situ hybridization.
In Situ Hybridization
In situ hybridization was performed as previously
described.13 Tissue sections were incubated overnight in
hybridization mixture containing digoxigenin-labeled, CVB3-specific
antisense riboprobes. Blocking with 2% lamb serum followed
posthybridization washing. Hybridized riboprobe was detected with a
sheep antidigoxigenin polyclonal antibody conjugated to alkaline
phosphatase (Boehringer Mannheim PQ). Slides were developed in
Sigma-Fast nitroblue tetrazolium-BCIP
(5-bromo-4-chloro-3-indolylphosphate tuluidinium) (Sigma),
counterstained in fresh carmalum, and examined for reaction product
by light microscopy.
cDNA Probes
Mouse hearts from each experimental group were pooled,
flash-frozen in liquid nitrogen, pooled, and stored at -70°C. mRNA
for each group was extracted and twice selected by oligo(dT)
chromatography. Fluorescently labeled cDNA
probes were generated by reverse transcription of poly(A)+ mRNA in the
presence of Cy3 or Cy5 dCTP (Amersham).
DNA Microarray
Microarrays contained 7000 cDNA clones randomly collected
from a normalized male Wistar rat heart cDNA library.14
Microarray fabrication and hybridization were performed at Incyte
Pharmaceuticals.15 cDNA inserts were generated by PCR
amplification with primers derived from flanking vector
sequences.16 PCR products were arrayed from 96-well
microtiter plates onto silanated microscope slides in an area of 1.8
cm2 using print tips driven by high-speed
robotics. Printed arrays were incubated for 4 hours in a humid chamber
and rinsed once in 0.2% SDS (1 minute), twice in
H2O (1 minute), and once in sodium borohydride
solution (1.9 g of NaBH4 dissolved in 300 mL of
PBS and 100 mL of 100% ethanol; 5 minutes). The arrays were submerged
in H2O (2 minutes) at 95°C, transferred quickly
into 0.2% SDS (1 minute), rinsed twice in H2O,
air dried, and stored in the dark at 25°C.17 Each pair
of fluorescently labeled cDNA sample probes was applied to the
microarray and allowed to hybridize competitively to the 7000 elements.
Degree of hybridization was quantified by sequential excitation of the
2 fluorophores with a scanning laser. Differential expression values
were represented as a ratio of intensities. Expression data
were omitted if the signal was <2.5-fold over local background or
derived from <40% of the area of the printed spot.
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Viral Time Course in the Heart
In situ hybridization using antisense CVB3-specific riboprobes was performed on myocardial sections from mice at each time point to determine the viral load, infected cell type, and infection variability among animals. At day 3 after infection, viral RNA is detectable in the myocardium of all animals. This amount of viral RNA is typically increased by day 9 after infection and completely absent by day 30 after infection, when no viral RNA is detectable in the myocardium of infected animals (Figure 1
).
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The characteristic localization of viral RNA within the myocardium at days 3 and 9 after infection exhibits a clear lack of preference for infection of endothelial cells, either those lining the ventricular cavities or those in small arteries, veins, or lymphatics within the tissue. In addition, there is no detectable infection of interstitial cells, such as fibroblasts. Indeed, the most visibly and clearly infected cell type is the cardiac myocyte. The range of cellular dynamics in the cardiac myocyte is reflected by the coexistence of cardiac muscle cells with or without a high-titer viral replication, but which have undergone cytopathic effects and may be resisting death in a fashion distinct from that of neighboring coagulated cells on the one hand and neighboring uninfected or "healthy" cells on the other. Additionally, infectious involvement of the ventricular septum and the left and right ventricular free walls appears generally similar in pattern, whereas atrial tissue has a more limited presence of viral genome in comparison.
Examination of contiguous sections stained with in situ hybridization
for viral RNA, hematoxylin and eosin, and Masson’s trichrome (Figure 2) illustrates that infected myocytes are
undergoing virus-induced cell death without immune involvement.
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Microarray Analysis of Gene Expression
DNA microarrays were used to evaluate changes in gene expression
in the myocardium at 3, 9, and 30 days after infection.
Approximately 7000 heart cDNA clones representing 4200
distinct genes were printed as high-density arrays. Arrays were probed
in duplicate with fluorescently labeled probes generated from
mRNA extracted from infected and control myocardial tissue at each time
point. Duplicate results were averaged, clones with discordant or
absent results were discarded entirely, and remaining clones increased
or decreased by 1.8-fold relative to control were considered
differentially expressed. A total of 619 cDNA clones were
differentially expressed in CVB3-infected myocardium. cDNA
clones that had no significant match, or matched only to an expressed
sequence tag in GenBank (http://www.ncbi.nlm.nih.gov/), were not
considered further. In total, differentially expressed cDNA sequences
corresponded to 169 individual known genes Figure 3.
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In a well-established model of viral infection, with consequent myocyte cell death, myocarditis, and reparative heart disease, and with a microarray containing a random and substantial repertoire of known sequences, the temporal changes in gene expression in a single target organ system were examined. Table 1 online (available at http://www. circresaha.org) summarizes 85 of the 169 known gene sequences found to be differentially expressed in this analysis in the context of their expression trends at each of the 3 postinfection time points.
Virus-Mediated Changes
Early events in CVB3 infection of susceptible mice have been
postulated to include gene regulation directly related to viral
replication in the host cell. In this study, the upregulation of
poly(A) binding protein (PABP), ubiquitin-specific protease UBP41, and
inorganic pyrophosphatase genes appear to support this concept. PABP
transcript upregulation is seemingly compensatory for viral degradation
of the PABP, which is cleaved by the CVB3 viral genome–encoded
protease 2Apro.18 PABP is thought to
play an important role in mediating the translation of
eukaryotic genes through facilitating the binding poly(A)
tails of different mRNA, thereby creating highly ordered protein-RNA
structures.19 Surviving cells may require an elevated
level of PABP to increase translation of other cellular proteins that
facilitate myocyte repair and the restoration of normal cardiac muscle
cell activity and integrity.
In addition to PABP, the gene of another protein involved in ribonucleoprotein complexes, heterogeneous nuclear ribonucleoprotein (hnRNP), was found to be upregulated at days 3 and 9 in our model of viral myocarditis. hnRNP may significantly impact gene expression by modulating the accessibility and/or interactions of trans-acting factors with particular DNA/RNA sequence elements and has been implicated in the translational inhibition of certain viral mRNA’s.20
The expression of other host genes modulated either through viral action or by host reaction involves a "struggle" between the expression of proapoptotic and antiapoptotic molecules. There is normally a balance between apoptotic and antiapoptotic signals, and cell death occurs in response to a persistent shift in this balance. Although the expressions of various apoptotic and antiapoptotic signaling molecules were differentially regulated in our study, their potential diverse and unknown functions make it impossible, in many instances, to make strong conclusions regarding the impact of their transcriptional regulation.
Of note, however, is the upregulation of the peripheral-type benzodiazepine receptor (PBR) gene on days 3 and 9 after infection, which appears to contribute to cell survival. The PBR localizes to the mitochondrial membrane, where it has been found physically associated with a voltage-dependent anion channel and is a component of the mitochondrial permeability transition pore complex (PT) formed at the contact site between the mitochondrial inner and outer membranes.21 Because the PT is known to be involved in apoptosis signaling22 and opening of the PT and voltage-dependent anion channel pores are regulated by Bcl-2 and Bcl-XL,23 it is likely that PBR is a critical component in an antiapoptotic process. This is supported by the wealth of PBR agonists that are potent antiapoptotic compounds.21 Thus, upregulation of the PBR transcript at days 3 and 9 may represent a host stress response aimed at cell survival.
Host Defense Response
There is increasing evidence that viral infection generates
reactive oxygen species24 and oxygen free radical
scavengers protect against virus-induced myocarditis.25
Thus, it is of interest that host responses to CVB3 infection include
increased expression of oxygen free radical scavenger genes concordant
with the timing of a strong, early reactive oxygen species presence and
stress response that diminishes as the remodeling process evolves.
These scavenger genes include glutathione peroxidase,
metallothionein, and thioredoxin, all of which
convey protection to cardiac myocytes from reactive oxygen
species.25 26 27
Further response to reactive oxygen species in this model was seen with the day 30 downregulation of the phosphotidylethanolamine N-methyltransferase gene, the protein of which is necessary for maintenance of membrane permeability integrity and is decreased in cardiac subcellular fractions by excess oxygen free radicals. Phosphotidylethanolamine N-methyltransferase activity has been found to be decreased in diseased hearts wherein oxygen free radicals are suspected to play a role in the pathogenesis of cardiac dysfunction.28 Correspondingly, the gene expression of another enzyme involved in the recycling of phospholipids,29 lysophospholipase, was found to be downregulated at days 3 and 9. These findings are intriguing, because mitochondrial membrane phospholipid composition may impact electron transport chain enzyme membership and cellular respiration.30 Such changes are supported by our model and are described in other models of cardiac dysfunction.31 32
Changes in Host Metabolism and Contractility
Typically, a switch of the chief myocardial energy source from
fatty acid ß-oxidation to glycolysis is observed both in
hypertrophy and in the failing heart. These changes were
highlighted by human patients with dilated
cardiomyopathy wherein enzymes in the mitochondrial
fatty acid oxidation cycle are downregulated.33
Furthermore, downregulation of the expression of the medium-chain
acyl–coenzyme A dehydrogenase gene, a key fatty acid ß-oxidation
enzyme, has been demonstrated during the progression from cardiac
hypertrophy to ventricular
dysfunction.33 In our model, the observed downregulation
of genes encoding enzymes apparently involved in the ß-oxidation of
saturated fatty acids and metabolic mitochondrial enzymes
involved in the citric acid cycle and electron transport chain
agrees with other models of cardiac dysfunction. In addition,
transcripts encoded by the mitochondrial genome, namely cytochrome
b, the mutants of which have been found to include cardiac
conduction block phenotypes,34 and 12S and
16S rRNA genes, the mutants of which have been reported with
hypertrophic cardiomyopathy,35 were
found to be downregulated on days 3, 9, and 30, respectively. Because
mitochondrial mutations have been implicated in both hypertrophic and
dilated cardiomyopathies,35 it is
tempting to speculate that downregulation of these and other
mitochondrial transcripts may be responsible for the loss of
function/cardiomyopathy observed in our model.
In parallel to myocardial energy source switching from ß-oxidation of
fatty acids to glycolysis, a reversion to the fetal energy substrate
preference pattern and the regulation of other
"hypertrophy" genes in the myocarditis model may be
important. Myocardial hypertrophy is often an early
hallmark of heart failure, occurring in response to a variety of
stimuli, including pathologic factors. The hypertrophic response occurs
as the heart attempts adaptation to increased demands on individual
myocytes for cardiac work. Our model would satisfy such a setting, as
noninjured myocytes compensate for injured and dead myocytes. In most
forms of cardiac hypertrophy, there is an increase in the
expression of embryonic genes, including the genes for
natriuretic peptides and fetal contractile
proteins.36 This re-expression of fetal isoforms mimics
the mitogenic response of many terminally differentiated
cell types, such as cardiomyocytes. Such responses were
seen in our model with the upregulation of genes encoding B-type
natriuretic peptide, atrial natriuretic factor,
ß-actin, and 
-actin.
Changes in ECM Genes
Genetically modified mice have also implicated ECM proteins in the
pathogenesis of heart disease. Both disruption of desmin, a component
of muscle adherens junctions in mice, and an ECM protein defect in
Syrian hamsters lead to cardiomyopathy, implicating
a link between myocardial ECM and the pathogenesis of
cardiomyopathy.36 In our model, a
large number of genes encoding ECM proteins and ECM/cytoskeletal linker
proteins were upregulated during the course of disease, beginning as
early as day 3. Overexpression of these genes not only corresponds to
an observed increase in ECM seen in the day 30 postinfection
myocarditic phenotype but also suggests an active reparative
and remodeling effort in response to myocardial damage and cell
loss.
Conclusions
Gene regulatory phenomena observed in our study provide a new
framework for understanding the initiation and progression of viral
myocarditis, including several new and potentially important
discoveries. Figure 1
online (available at http://www.circresaha.org)
summarizes several of the important regulatory events and places a
microenvironmental and temporal context for their expressive changes.
Our approach to this study was based on a powerful molecular tool in an
unbiased assessment of differential gene regulation. The inter-rodent
sequence similarities and homologies in many genes
(ftp://devftp.informatics.jax.org/pub/informatics/reports/index.html;
http://www.latrobe.edu.au/www/genetics/reports.html) made it feasible
to use the carefully prepared rat cDNA library. We used this library as
our collaborators were studying rat models of heart disease with
preprepared rat chips, and no comparable ability to screen against a
mouse library or develop a rat model of viral myocarditis was
available. It should be noted, however, that qualitatively the
hybridization of the 2 rodent mRNAs were found to be equivalent,
indicating no quantitative species-specific variation, and the
mouse-rat hybridization demonstrated higher quality than the rat-rat
hybridization with respect to hybridization specificity. Furthermore,
the relative labeling intensity for any given gene across these 2
species was identical, indicating that under our stringency conditions,
no loss in sensitivity occurred. Finally, and perhaps unexpectedly, a
consistently higher signal-to-noise ratio was observed with the
hybridization of mouse cDNA in comparison with rat cDNA against the rat
chip. The authors have no explanation for this except to surmise that
perhaps background labeling of the cDNA spots includes hybridization to
species-specific repeats (the Alu repeats, for example) found not only
in the genome but also in the 3', untranslated end of the mRNA strands.
If so, cDNA made from mouse mRNA might show less complementary binding
to the Alu-type repeats found in the rat cDNA spots and demonstrate
less background binding. Nevertheless, an additional species-specific
approach could possibly yield additional insights regarding the
regulation of viral receptors, mouse-specific cytokines, and
other species-specific gene expression, yet the richness of our
observations thus far has provided many potentially important new
opportunities to understand the pathogenesis of enteroviral heart
disease.
This initial portrait of an in vivo model of human disease is considered a first step, inasmuch as microarray results and trends are limited to a portion of the total regulatory picture in cellular processes. In addition, the demonstration and exploration of transcriptionally regulated events using expression microarray technologies, while qualitative, is only quantitative in a relative fashion.12 Furthermore, the process involves a mixed, interactive cell population for origin of the probe transcripts in our model tissue. The mixed population of cells used for probe construction not only contained many different cell types including infected myocytes, but also noninfected myocytes with infected neighbors in close proximity, releasing stimuli or signaling factors (autocrine or paracrine). Thus, gene regulation observed in these whole-heart gene expression assays was due to a composite portrait rather than sole regulation of myocyte gene expression in response to viral infection. Although not unicellular, our approach does capture the entire pathophysiological microenvironment associated with the myocarditic disease process and as such may provide directions of greater pertinence to the pathogenesis and ultimate treatment of viral myocarditis. Our observations do not exclude the possible influence of systemic factors, originating from other organs and tissues, on the gene expression profile of the myocardium.
Future Directions
The value of additional studies that assess gene expression
profiles of individual cells or cell populations are, of course,
appreciated.37 However, the strengths and limitations of
either approach necessitate gleaning insights from both. Laser capture
microdissection or laser pressure catapult will in part resolve the
"whole-organ" issue, recognizing the limits in terms of members of
a given cell type (ie, myocytes) that can be effectively evaluated in
an experiment designed for purposes such as
ours.38 39 40
Additionally, these studies on gene expression changes in mice after CVB3 infection will be expanded by our group and others to include both more time points and additional comparisons such as myocarditis susceptible versus resistant and immunocompromised versus immunocompetent mouse hearts. Together, these integrated approaches toward characterizing gene regulation in the murine model of CVB3-induced myocarditis will both further present understanding and generate numerous leads regarding investigations into genetic governance of susceptibility, pathogenesis, and progression. Furthermore, these studies will lead to heightened understanding of myocardial viral infection in specific and heart dysfunction in general and may direct our attention to potential therapeutic targets for both.
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Acknowledgments |
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We thank the British Columbia Health Research Foundation (20R-52234), Medical Research Council (20R-90214), and Heart and Stroke Foundation of British Columbia and Yukon (20R-53837) for grant-in-aid support. Dr Michael Allard provided helpful critique and advice in the preparation of the manuscript. We also thank the following people for their valuable contributions to the experiments and manuscript preparation: Deborah Damm, Andrew Lam, Lisa Garrard, Paul Cheung, David Granville, and Zongshu Luo.
Received March 10, 2000; revision received June 19, 2000; accepted June 20, 2000.
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