JACC Vol. 9, No.6June 1987:1311-9

EXPERIMENTAL STUDIES

Coxsackievirus B3 Murine Myocarditis: A Pathologic Spectrum ofMyocarditis in Genetically Defined Inbred Strains

AHVIE HERSKOWITZ, MD,* LUANNE J. WOLFGRAM, PHD,t NOEL R. ROSE, MD, PHD,*·t

KIRK W. BEISEL, PHDt

Baltimore, Maryland

1311

Group B coxsackieviruses are the most frequent caus­ative agents in human viral myocarditis. Susceptibilityto viral infections varies widely among individuals. Inthe mouse, coxsackievirus B3 also causes myocarditis.The differential susceptibility of different inbred strainsof mice to coxsackieB3-induced myocarditis also appearsto be under genetic control. This study details the histo­pathology of coxsackie B3 myocarditis in six differentinbred strains of micefor the first 45 days after coxsackieB3 infection. These strains differ either in the haplotypesof their major histocompatibility complex or in theirbackground genome.

During the first 7 days after coxsackie B3 infection,there are dramatic differences among strains with re­spect to prevalence and severity of myocarditis. Focalzones of myocyte necrosis involving polymorphonuclear

In humans the group B coxsackieviruses have been identifiedas the viral agents most frequently associated with viralmyocarditis and pericarditis (1-5). The pioneering studiesof Lerner and Wilson (5) have given us a model for thestudy of virus-induced heart muscle disease. Their studiesdemonstrated that 14 day old mice, inoculated intraperito­neally with coxsackievirus B3 , develop severe inflammatoryheart muscle disease. The lesions observed in coxsackie Brinfected mice are histologically similar to those in humans(6-12). Because susceptibility of different individuals toviral infections varies widely, we postulated that suscepti-

From the *Departments of Medicine and of tImmunology & InfectiousDiseases, the Schools of Medicine and of Hygiene and Public Health, TheJohns Hopkins University, Baltimore, Maryland. This work was supportedby Public Health Service (PHS) Grants HL-27932, HL-30144. CA-34202and Training Grant 5T32-HL07227 from the National Heart, Lung andBlood Institute and the National Cancer Institute, respectively, NationalInstitutes of Health, Bethesda, Maryland. Computational assistance wasreceived from CLINFO, sponsored by National Institutes of Health GrantRR-00035.

Manuscript received April 22, 1986; revised manuscript received Oc­tober I, 1986, accepted October 22, 1986.

Address for reprints: Ahvie Herskowitz, MD, Cardiology Division,The Johns Hopkins Hospital, 600 North Wolfe Street, Carnegie 568, Bal­timore, Maryland 21205.

© 1987 by the American College of Cardiology

leukocytes as well as contraction band injury appear tobe the early manifestations of direct viral injury. Fourof the six strains, though, continue to show myocardialinflammation after day 9. This late phase myocarditis ischaracterized by the emergence of mononuclear cellswithin healing foci of myocyte necrosis as well as a dis­tinctive diffuse interstitial pattern of myocarditis. Thestrains that develop this late ongoing myocardial inflam­mation frequently produce heart-specific autoantibod­ies.

Thus 1) the pathologic features of murine coxsackieB3 myocarditis change over the course of the illness, and2) genetic susceptibility to both early and late phasemyocarditis differs markedly among various mousestrains.

(J Am Coil CQrdioI1987;9:131l-9)

bility of different inbred strains of mice to coxsackie Brinduced myocarditis would differ in accordance with theirrespective genetic constitutions. In a previous time coursestudy (12), a number of genetically defined mouse strainswere found to vary widely in the' extent and duration ofviremia, in the temporal appearance and titer of neutralizingantibody and in the prevalence of myocarditis. These in­vestigations supported the hypothesis that there is geneticregulation of susceptibility to coxsackie Bj-induced disease.

This study will detail the histopathology of coxsackie B3

myocarditis during the first 45 days after infection. Sixstrains of mice that differ either in the haplotypes of themajor histocompatibility complex or in their backgroundgenome (that is, nonmajor histocompatibility complex genes)are the subject of this report. The observed strain differencesin the severity of myocarditis as judged by the quantity ofdamaged myocardium, as well as differences in histologicpatterns of myocardial injury, further indicate genetic con­trol of susceptibility to coxsackie B3 myocarditis. The pres­ent study also documents marked histopathologic and straindifferences between early and late phase myocarditis andtherefore supports the concept that early direct virus-inducedinflammation differs from a later immunopathologic myo-

0735-1097/87/$3.50

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Table I. Prevalence and Severity of Focal Myocardial Lesions and Contraction Band Necrosis in Early Phase Myocarditis

Focal Lesions Contraction Band Necrosis Mean %

Day 5 Day 7 Day 5 Day 7Myocardium

AffectedStrain Group A B A B A B A B (days 5 and 7)

A.BY IA 100(9) 3.3 :!: 0.6* 100(8) 2.1 :!: 0.4 100 4.3 :!: 1.0* 78 1.0 :!: 0.8 5.5 :!: LOtA.SW lA 92(11) 2.7 :!: 0.5* 100(11) 5.3 :!: 0.9* 92 3.2 :!: 0.8* 63 1.0 :!: 0.5 6.4 :!: 0.7tA.CA IB 28(7) 0.1 :!: 0.1 50(4) 0.4 ::!: 0.1 0 0 0 0 0.5 ::!: 0.1C3H.NB IB 55(9) 1.3:!:1.2 63(8) 4.4 ::!: 2.3 11 0.5 ::!: 0.5 25 0.8 ::!: 0.5 4.2 ::!: 1.8

BIO.PL 2 89(9) 0.6 ::!: 0.2 58(12) 4.0 ::!: 1.4 0 0 0 0 2.0 ::!: 0.7

BI0.S 2 91(11) 0.4 :!: 0.1 67(9) 1.6 ::!: 1.0 0 0 0 0 0.9 ± 0.4

ANOYA P < 0.001 P = 0.013 P = 0.04 NS P < 0.001

*Different from all other groups (Neuman-Keuls multiple range test); tdifferent from A.CA, B IO.PL and B IO.S (Neuman-Keuls multiple range test).Column A indicates the percent of animals showing this form of pathologic involvement; column B indicates the percent of myocardial involvement :!:SE only in affected animals. Numbers in parentheses indicate the number of animals analyzed for both focal lesions and contraction band necrosis.

carditis. The susceptibility to both early and late myocarditisappears to be under multigenic control.

MethodsAnimals. Fourteen day old A.BY/SnJ and A.SW/SnJ

female mice were purchased from Jackson Laboratories. Allother female animals of the same age from the A.CNSnJ,C3H.NB/SnJ, BIO.S/SgSf and BIO.PUSgSf strains wereraised in our own animal facilities. Both the uninfected andinfected animals were kept with their mothers or foster mothersuntil they reached 4 weeks of age. The coxsackie Bj-infectedmice were housed in a separate room.

Tissue culture. Vero monkey kidney cells were obtainedfrom Flow Laboratories. The cells were maintained in min­imal essential medium from Gibeo, which was supple­mented with 10% fetal calf serum, glutamine, antibioticsand N-2-hydroxyethylpiperazine-N' -2-ethane sulfonic acid(HEPES) buffer. The cells were incubated at 37°C in ahumidified atmosphere containing 5% carbon dioxide. Whenused in an assay for virus, the cells were diluted in medium

containing 2% fetal calf serum and then plated into half­area (N2) 96 well microtiter plates (Costar).

Virus preparation and titration. Coxsackievirus B3

(Nancy strain), originally obtained from A.M. Lerner, WayneState University, was maintained in our laboratory. A stockculture of virus was prepared by plating a I :200 dilution ofcoxsackie B3 onto confluent cultures of Vero monkey kidneycells. The flasks were allowed to incubate at 37°C for 24hours or until a 4 + cytopathic effect was seen. At this timethe remaining adherent cells were scraped off and the re­sulting suspension underwent three freeze-thaw cycles. Theviral suspensions were then centrifuged and the supernatantwas divided into aliquots and stored at -70°C until use.The virus pool was subsequently titrated in triplicate wellson microtiter plates containing Vero cells. After incubationfor 3 days at 37°C, the median tissue culture dose (TCID50)

was determined by using the method of Reed and Muench.Mouse hearts were also analyzed for virus content usingthis titration procedure.

Infection and necropsy. Two week old female mice ofeach of the six strains (approximately IO/group) were in-

Table 2. Prevalence and Severity of Myocardial Lesions and Prevalence of Interstitial Inflammation in Late Phase Myocarditis

Mean %

Day 9 Day 15 Day 21 Day 45Myocardium % Prevalence of

Affected lntersiitial

Strain A B A B A B A B (days 9 to 45) lnflarnmationt

A.BY 100(4) 2.6 ::!: 1.0 100(7) 14.6 ::!: 4.7* 75(8) 2.8 :!: 0.7 106(4) 2.6 :!: 1.4 6.7 :!: 2.lt 26

A.SW 92(12) 6.2 ::!: 2.4 88(8) 5.6 ::!: 2.5 80(10) 6.0:!: 2.1 60(12) 2.6 :!: 0.8 6.0 ::!: 1.3t 6O§

A.CA 78(9) 2.0 ::!: 1.2 62(8) 0.3 ::!: 0.2 73(11) 4.1 :!: 2.2 77(13) 1.6 :!: 0.8 3.2 ::!: I.3t 13

C3H.NB 86(7) 12.5 ::!: 5.7 55(11) 4.0 ::!: 2.3 70(10) 5.6 :!: 2.2 64(11) 3.1 :!: 1.0 7.5 ::!: 2.0t 20

BIO.PL 80(10) 2.4 :!: 1.2 70(10) 1.1 :!: 0.8 36(11) 0.6 ::!: 0.3 22(13) 0.3 :!: 0.2 1.\ ::!: 0.4 0

BIO.S 50(10) I.I :!: 0.7 38(8) 0.5 :!: 1.5 20(10) 0.2 :!: 0.3 20(10) 0.3 :!: 0.2 0.7 :!: 0.4 0

ANOVA NS P = 0.002 NS NS P < 0.001 P < 0.01

*Different from all other groups (Neuman-Keuls multiple range test); tdifferent from B IO.PL and BIO.S (Neurnan-Keuls multiple range test); :!:percentof animals affected by focal lesions involving> 1% myocardium; §different from all other strains (chi-square analysis). Column A indicates the percentof animals showing focal myocardial lesions; column B indicates the percent of myocardium affected by focal lesions ± SE only in affected animals.Numbers in parentheses indicate the number of animals.

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removed and stored at -70°C for viral analysis. The re­mainder of the heart was fixed in formalin for histologicpreparation. The heart weights were also determined, sothat heart to body weight ratios could be calculated. Animalsthat died during the study were not included in the analysis.

Histology. Hearts were fixed in 10% buffered formalinand embedded in paraffin according to standard procedures.Transverse sections were made at two levels to ensure ad­equate representation of the pathologic features. An addi­tional section with atria, pulmonary and aortic roots wasobtained when the heart was large enough to obtain the twostandard myocardial sections. Ten serial sections, each 5/Lm thick, were made at each level and stained with he­matoxylin-eosin. Representative cases were treated withMasson's trichome and von Kossa's stain for calcium.

Microscopy. Two cardiac pathologists reviewed all caseswithout knowledge of other data and initially graded themyocardial pathologic findings using a subjective gradingsystem (grades 0 to 4) described by EI-Khatib et al. (13).To better assess the amount of myocardium affected in eachmouse a quantitative assessment of parenchymal lesions wasperformed by one of the pathologists (AH). The secondserial section from each case was placed under 25 x mag­nification using a light microscope with direct video inter­face with a Zeiss Videoplan image analysis microcomputer.For each heart, total left and right ventricular areas and areaof myocardial necrosis were determined by serial recon­struction of integrated cross-sectional areas of the histologicsections. This was done by first contouring the left and rightventricular epicardial and endocardial surfaces. Zones ofmyocardial necrosis were then contoured and the specifiedcross-sectional areas were automatically calculated. The to­tal area of myocardium composed of focal necrotic lesionswas then expressed as a percent of the total myocardial areameasured; at days 5 and 7 the total area of myocardiumcontaining contraction band necrosis was also expressed asa percent of the total myocardial area measured. Repeatedmeasurements yield consistent results with only rare in­stances approaching a 10% error in specimens with smallfoci of involvement.

Mice were considered to have myocarditis if any formof inflammation or necrosis was present (Tables J and 2).To distinguish cases with only one microscopic focus ofnecrosis from other cases with more significant involve­ment, an arbitrary cutoff of I% of myocardial involvementwas used to define affected cases in Figure I.

Interstitial inflammation was considered to be presentwhen clusters of round, mononuclear cells (>5/high powerfield) confined to the interstitial space between nonnecroticmyocytes were found. This could be either a focal or amultifocal finding. Care was taken to strictly define con­traction band injury. In addition to a focal, dense clusteringof intracellular hypereosinophilia, loss of myofibrillar def­inition was required for cells to be considered affected.

33

810.5

C3H.N8A.CA

31

B10.PL810.5

C3H.NB0

A.CA

50 GROUP 2

Ul 40-l<t::I:HZ 30<t

U.0

a: 20UJ[]J 13::I:~ 10z

50 GROUP 1A

.-----EARLY----, ,....--LATE----,

Ul 40-l<t::I:H 18Z 30<t

U.0

a: 20UJ[]J::I:~ 10z

0A.BY A.5W A.BY A.5W

50 GROUP 18

Ul 40-l<t::I:HZ 30<t 27 22

U.0

a: 20UJ[]J::I:~ 10z

B10.PL0 .........---

Figure 1. Numbers of involved (affecting> I % of the myocar­dium) versus uninvolved mice when time periods are dividedintoearly (days 5 to 7) and late (days 9 to 45) phases. Hatched bars,number of animals with myocarditic lesions > I % myocardium;open bars, number of animals without myocarditic lesions orlesions <1%.

jected intraperitoneally with 0.1 ml containing lOS TCID socoxsackie B3 , and groups of animals were killed at 2, 3, 5,7, 9, 15, 21 and 45 days after inoculation. The number ofmice per sacrifice is given in Tables I and 2. An equalnumber of control animals received an uninfected Vero celllysate preparation that was diluted in the same fashion asthe coxsackievirus B3 inoculum. After being weighed, themice were killed by retroorbital bleeding. The mouse serawere individually collected and stored at - 70°C for as­sessment of viral and antibody content. Heart apex was also

1314 HERSKOWITZ ET AL.COXSACKIEVIRUS B3 MURINE MYOCARDITIS

lACC Vol. 9, No.6June 1987:1311-9

--l«>H

>a::::>(/l

~

ZWua:wa..

100

75

50

25

- ..::-....:-;._~__~ - - - - - - - - - - - - - - - A...:,.C,A

_001 ~~~ :~~_OOj B10 .S

,·········'··L1..1.1. .

--------_.o.o.oo. oo••_ooo.__.oo •..o iLoS.~

A.BY

Figure2. Survival curves for Group IAstrains (A.SW and A.BY) over the first45days after coxsackie B3 infection. A.BYhas a significantly greater mortality ratethan A.SW during the first 9 days (p <0.(01). Group IB strains are A.CA andC3H.NB and Group 2 strains are BIO.PLand B10.S.

O-r-----t---t---+----------------!5 10 15

ELAPSED DAYS45

Statistical analysis. Differences among the six strainsin prevalence and severity of lesions, heart viral titers andinterstitial inflammation weredetermined by analysis of var­iance testing using Neuman-Keuls multiple range test andchi-square analysis. Mortality data were analyzed compar­ing life tables using the generalized Wilcoxon test. Differ­ences between the severity of focal lesions and contractionband necrosis in early phase myocarditis were determinedby one-tailed t test.

Experimental plan. Previous studies suggested thathostsusceptibility to coxsackie Bj-induced myocarditis is deter­mined by multiple genes (12). To analyze the effect of thisgenetic regulation on the expression of coxsackie Bj-in­duced myocarditis we chosesix strainsfor detailedanalysis.These strains were selected to provide examples of strainsthat differ both in genes within the H-2 complex (majorhistocompatibility complex) and in the background genes(nonmajor histocompatibility complex). The six strains areA.BY (H-2 b

) , A.CA (H-2 f) , A.SW (H-2 S

) , BIO.S (H-2 S) ,

BIO.PL (H-2U) and C3H.NB (H-2P) . The A strainH-2 con­

genies share the same background genes, but differ in their

major histocompatibility complex genome (H-2 b, H-2s or

H-2 f) . The B10 congenics also share the same background

genes but differ in haplotypes (H -2S or H-2 U) . In contrast,

the A.SW and BIO.S share the same major histocompati­bilitycomplexgenes (H-2 S

) but differ in background genes.Two week old mice from each strain were infected withcoxsackie B3 and sacrificed at days 2,3,5,7,9,15,21 and 45after coxsackie B3 infection. Results are assessed in termsof mortality, prevalence and severity of myocardial lesionsas well as histologic patterns of injury.

ResultsMortality (Fig. 2)

Only mice of the A.SW and A.BY strains died duringthe 45 day study period. None of the control animals givenuninfected cell lysate died. Mice of A.BY strain had sig­nificantly more deaths than did those of the A.SW strainthrough day 9 (p < 0.(01), at which time the rates ofmortality no longerdifferedbetweenthe two strains. At day

Table 3. Summary Data of Immunologic Profiles of Responses to Coxsackievirus B3 Infection

NeutralizingViremia Antibody Autoantibody

Strain Group Mortality Day 2 Day 3 Production

A.BY IA + High* Absent PresentA.SW IA + High Absent PresentA.CA IB Low Present PresentC3H.NB 1B Moderate] Present PresentBIO.PL 2 Lowl Present AbsentBIO.S 2 Moderate Present Absent

*range2.75 to 4.5 (10"/0.1 ml); trange 1.25 to 3.25 (10"/0.1 ml); lrange 0.75 to 2.6 (10"/0.1 ml). (Modifiedfrom Wolfgram U et al. [121.)

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9 there was a 43% mortality rate among mice of the A.BYstrain and a 27% mortality rate among those of the A.SWstrain. After day 15, only sporadic deaths were noted inmice of either strain.

Histopathology of Coxsackievirus B3 Myocarditis

On the basis of previously reported data (12) summarizedin Table 3, mice from the six strains examined were placedinto two broad groups. Mice from all six strains developedacute myocarditis 5 days after virus inoculation. Mice fromstrains in Group I (A.SW, A.BY, A.CA and C3H.NB)proceeded to develop an ongoing myocarditis as well, whereasthose from strains in Group 2 (BlO.S and BlO.PL) did not.Group 1 strains were further divided into two subgroupsbased on the severity of early myocarditis. Group lA (A.SWand A.BY) strains comprised the most severely affectedstrains as judged by early mortality and high prevalence ofearly myocarditis (Fig. 1 and 2). Group IB (A.CA andC3H.NB) had no early mortality and a more variable earlyprevalence of myocarditis.

Early (days 5 and 7) phase myocarditis. The earliesthistologic abnormalities could be appreciated by day 5, atwhich time no gross abnormalities were present. Two typesof lesions could be seen in early phase myocarditis. I) Focalzones of necrosis were characterized by calcified clustersof necrotic myocytes found randomly distributed throughoutthe right and left ventricular walls (Fig. 3A and B). 2)Individual small clusters of myocytes containing dense con­traction bands (Fig. 3C) could also be identified in the mostseverely affected mice. Contraction band necrosis was fre­quently not accompanied by inflammatory cells during thisearly time period and may therefore have represented anearly yet nonspecific light microscopic abnormality in thismodel of myocarditis. Vascular and perivascular inflam­mation was rare and was not a prominent abnormality inany of the specimens. Interstitial inflammation was not pres­ent at these two time points.

Late (days 9 to 45) phase myocarditis. Mice in Group2 did not show evidence of ongoing inflammation after day7. The lesions seen at days 5 and 7 were replaced by denselinear bands of connective tissue dividing individual musclebundles (Fig. 4A). Fibrous scars were primarily composedof fibrocytes, with only rare mononuclear cells seen.

Mice in Groups lA and IB, in contrast, showed, afterday 7 ongoing inflammation consisting of focal lesions thatbecame heavily calcified, quite discrete and large (Fig. 4B).Inflammatory cells within the lesions were abundant andconsisted of plump mononuclear cells, as well as a newpopulation of smaller, round mononuclear cells (Fig. 4C).A prominent interstitial cellular infiltrate, composed of bothlarge and small mononuclear cells, also appeared for thefirst time (Fig. 40). Control mice did not show any inter­stitial inflammation at any time period. By days 21 and 45,

Figure 3. Medium power photomicrographs on day 5. A, A.SWstrain. Focal myocyte necrosis is characterized by basophilic gran­ular degeneration of the myocyte. Polymorphonuclear leukocytesare seen engulfing cellular debris (arrows). Neighboring myocytesare normal. B, C3H.NB strain. Large mononuclear cells engulfcellular debris within a more mature, healed necrotic focus (smallarrows). An individual cell with contraction band injury is alsopresent (large arrow). C, A.BY strain. A cluster of myocardialcells is seen with dense contraction bands with focal loss of myo­fibrillar definition. A,B and C stained with hematoxylin-eosin; orig­inal magnification: A and B, x 550; C x 650; all reduced by 5%.

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the focal necrotic zones became less cellular and more fi­brotic (Fig. 4E) and only a few animals continued to haveinterstitial inflammation.

In summary. early injury was characteri zed by focal nec­rotizing lesions as well as contraction band necrosis. Activelate lesions were seen only in Groups IA and 1B and werecharacterized by the appearance of mononuclear cells bothwithin the enlarging focal lesions and in the interstitium as

Figure 4. Low and medium power photomicrographs , days 151021. A, B IO.PL strain. Healed lesions are characterized by linearbands of loose connecti ve tissue containing a sparse mononuclearcell infiltrate. B, C3H.NB strain . Densely calcified, discrete, focallesions containing a moderate inflammatory cell infiltrate. C, A.CAstrain. Healing focus of myocyte necrosis consisting of a mixedpopulat ion of large and small mononuclear cells. D, A.CA strain.Mononuclear cells arc seen within the interstitial space (a r rows)separating normal-appe aring myocytes. E, A.SW strain. Evolutionof a mature focus of myocyte damage which contains many mono­nuclear cells. Clusters of interstitial mononuclear cells surroundthe focus as well. A to E stained with hematoxylin-eosin; originalmagnifications: A, x 220, B, x 50, C to E, 550, all reduced by5%.

early as day 9 . Evidence of ongoing inflammation withinfocal lesions and mononuclear interstitial inflammation wasseen through day 21 but active inflammation was seen onlyin rare cases as late as day 45.

Severity of Myocardial Disease

Early phase myocarditis (days 5 to 7). Group IA strainswere characterized by the highest early prevalence of my­ocarditis as well as the most extensive total areas of myo-

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cardium affected by the myocarditic process at day 5 (p <0.00l) (Table I). The histopathologic features of early my­ocarditis, though, could be divided into two morphologicpatterns, In addition to the classicaily described focal nec­rotizing lesions seen in murine myocarditis (Fig. 3A andB), some severely affected mice had either focal or diffuseareas in which myocytes contained contraction band injury(Fig, 3C).

Mice from the A.CA strain (Group IB) and Group 2strains did not have contraction band necrosis at either day5 or 7, On day 5, contraction band necrosis accounted for56% of the total myocardial damage in Group I A mice(Table I). Contraction band necrosis was significantly greaterin Group lA animals as compared with those in Groups I Band 2 (p < 0.001), This form of injury was seen in oneseverely affected animal from the C3H.NB strain (GroupIB) and was not present in any Group 2 animals. By day7, contraction band necrosis accounted for only 32% of thedamaged myocardium in A.BY mice and for only 16% inboth the A.SW and the C3H.NB mice. The total myocardialarea affected by contraction band necrosis on day 5 in thesestrains was significantly greater than that on day 7 (p =

0.001 ), The switch from contraction band necrosis on day5 in severely affected mice to focal necrotizing lesions onday 7 was supported by a significant increase in the amountof myocardium affected by focal lesions on day 7 as com­pared with day 5 (p = 0.004) .

Therefore , contraction band necrosis in Group lA micewas the most significant early form of myocardial damageat day 5. These animals had more contraction band necrosisthan did mice froin ail other strains at this time. By day 7,focal lesions appeared more prominent and represented auniform histologic pattern of myocardial injury at day 7.This suggests that contraction band necrosis may representa severe form of early virus-induced myocardial damage insusceptible strains.

When the total area of myocardium damaged by bothfo cal lesions and contraction band necrosis was calculatedfor both days 5 and 7, mice from Group 1A strains weresignificantly more damaged than were those from the A.CA(Group IB) and Group 2 strains (p < 0.001). The total areaof myocardial damage from contraction band necrosis forboth days 5 and 7 was significantly increased in Group IAstrains as compared with ail other strains (p < 0.001).

The A,CA strain (Group 18) had both the lowest prev­alence of myocarditis at days 5 and the f ewest mice withlesions affecting more than 1% of the myocardium (Table1, Fig, 1), The histologic features were uniformly those offocal lesions with no contraction band injury seen. TheC3H.NB strain (Group IB), on the other hand, had anintermediate prevalence of early phase myocarditis that re­mained stable throughout the study period (Table I, Fig.1). The extent of myocarditic lesions in the C3H.NB strain

was comparable with that of the Group IA strains althoughearly contraction band injury was not a prominent finding.

In Group 2, the B IO.S and B IO.PL strains had a highearly prevalence of myocarditis but the extent of myocardialdamage was frequently smail and occupied > I % of themyocardium in only I I of 4 I animals (Table I , Fig. I).Group 2 strains exhibited only focal lesions and contractionband injury was not seen.

Late phase myocarditis (days 9 to 45). The histopath­ologic pattern of late phase myocarditis was remarkablysimilar in ail four strains constituting Groups IA and lB.Although the extent of myocardial damage quantitated inTable 2 reflects the quantity of myocardium composed offocal necrotizing lesions arranged in discrete clusters (Fig.4B), all four strains developed a distinctive interstitial mono­nuclear ceil infiltrate that was never seen in Group 2 strains(Table 2). This interstitial infiltratewas only seen in animalswith focal lesions affecting > 1% of the myocardium, al­though no direct correlation between the extent of myo­cardial necrosis and the prevalence of interstitial inflam­mation could bedemonstrated. Chi-square analysis revealeda significant difference (p < 0.01) with respect to prevalenceof interstitial inflammation among the four strains. Thisdifference is accounted for by the increased prevalence inthe A.SW strain.

Group 1A strains continued to have a high prevalenceof myocarditis as well as extensive myocardial involvementduring late phase myocarditis (Table 2, Fig. I). The A.BYstrain in particular had the highest prevalence of diseaseaffecting I% of the myocardium during the late time periods(days 9 to 45) (Fig. I ), as well as the most extensive myo­cardial damage at day 15 (Table 2).

Although Group J8 strains exhibited remarkable differ­ences in susceptibility to early phase myocarditis, both theprevalence of focal myocarditis and the extent of myocardialdamage were similar during days 9 to 45 (Table 2, Fig. I) .Indeed. there were no significant differences between theGroup IA and IB strains when the extent of myocardialdamage was expressed as a mean value over the entire periodfrom day 9 to day 45 (Table 2). Of interest is the A.CAstrain. This strain had a low early prevalence of myocarditisassociated with small focal lesions (Table I). Over the latetime period, though, the prevalence of myocarditis rose, thesize of the myocarditic lesions enlarged and the strain de­veloped scattered cases with interstitial inflammation (Table2, Fig. I).

Group 2 strains did not have ongoing myocardial ne­crosis or inflammation after day 7. Over the later time period(days 9 to 45) the prevalence of myocarditis declined dra­matically and few mice had significant amounts of myo­cardium damaged (Table 2, Fig. I). These strains could beconsidered the least susceptible strains to late phase myo­carditis.

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Discussion

Our previous studies (7,12,14) have emphasized a ge­netic basis for the variable prevalence of murine myocarditisseen after coxsackie B3 infection. The most effective andprotective immune response can best be identified in micein Group 2. Low levels of viremia, consistently low neu­tralizing antibody production and rapid clearance of virusfrom the blood are seen (12). A subdued inflammatory re­sponse rounds off the immunologic and histopathologic pic­ture that characterizes this group's resistance to coxsackieB3 infection. A profile of the most severely affected strains(Group l A), in contrast, is characterized by high levels ofviremia, delayed production of neutralizing antibody, de­layed viral clearance from blood and significant mortalityduring the first 15 days after infection. In addition, bothGroup lA and Group lB mice develop late interstitial my­ocarditis associated with autoantibody production. Our cur­rent study, therefore, indicates that genetic control of theseveral immune responses to coxsackievirus determines theseverity of the myocardial damage seen in surviving animalsduring both early and late postinfection periods. Indeed, thehistologic characteristics of this model of murine coxsackieB3 myocarditis appear to stress two important factors: 1)the genetic composition of the strain, and 2) the time periodafter coxsackie B3 inoculation that the heart tissue is beingexamined.

Strain differences in coxsackievirus 8 3 myocarditis.In our murine myocarditis model, dramatic strain differ­ences with respect to prevalence, extent and histologic pat­tern of myocarditis occur in both the early and the late phasesof the disease. The early histologic pattern of myocardialinflammation common to all strains consists of focal my­ocyte necrosis. The extensive contraction band injury inGroup lA strains on day 5 may be an early manifestationof a more severe form of early phase myocarditis. Thesedifferences in the light microscopic histologic patterns ofmyocarditis during this early time period, therefore, supportgenetic differences in the severity of the inflammatory re­sponse during early myocarditis. In addition, the variabilityof light microscopic features of late phase myocarditis in­dicates distinctive genetic predisposition toward late phasemyocarditis. After day 7, Group 2 strains no longer haveevidence of ongoing inflammation, whereas Group lA andIB strains continue to show active inflammation. This on­going inflammation is characterized not only by foci ofmyocyte necrosis but also by the emergence of a mono­nuclear interstitial infiltrate. Although no histopathologicdefinition of myocarditis in humans is universally accepted,most investigators agree that myocarditis is present whenthere is an interstitial infiltrate associated with myocyte ne­crosis. The histologic patterns of late phase myocarditis inour murine model are very similar to those described inmost cases of human myocarditis (6,8).

Genetic control of susceptibility to myocarditis. Theassociation between histocompatibility antigen types andidiopathic cardiomyopathy in humans has recently been sug­gested (15). The specific role of the major histocompatibilitycomplex and background genetic loci in our murine modelof myocarditis can best be illustrated by comparing strainsthat share specific loci. The A.SW (Group lA) and B IO.S(Group 2) strains share the same H-2s haplotype (majorhistocompatibility loci) so that it appears that the A back­ground genome may control both the severity of early phasemyocarditis and the emergence of the delayed onset mono­nuclear cell myocarditis. The difference in the A H-2 con­genies are best illustrated by comparing the A.CA (H-2 S

)

(Group IB) with either the A.SW (H-2 S) or A.BY (H-2b

)

strain (Group lA). The A.CA strain is the least affectedstrain at day 5 in both prevalence and severity of lesions(Table 3, Fig. 1). Yet, by day 9, when the late pattern ofhistologic change first appears, the A.CA strain is indistin­guishable from the other A H-2 congenics with similar back­ground genes, in both prevalence and extent of myocarditis.Gauntt et al. (16), using 14 mouse strains, also found thatthe severity of early phase myocarditis is affected by H-2and background genes.

The major histocompatibility complex. therefore, maycontrol the dampened early response to coxsackie B3 my­ocarditis, but the A background genome appears to controlthe characteristic late pathologic changes seen in all threeof the A H-2 congenics strains used in this study. Theimmune response may therefore have two paradoxic effectsduring the course of coxsackie B3 infection. The first is aprotective action that prevents continued viral replication.The second effect is the development of ongoing myocardialinflammation that coincides with the production of myo­cardial-specific autoantibodies (12). Whether these auto­antibodies indicate that late phase myocarditis is an autoim­mune disease or whether they are simply markers of ongoingmyocardial injury is not known at this time.

In humans, the relation between chronic myocarditis andautoimmunity has been suggested from studies describingthe development of heart-specific autoantibodies in patientswith suspected viral myocarditis (17), and the presence ofautoantibodies in surveys of patients with idiopathic dilatedcardiomyopathy (18). The heart muscle-specific autoanti­bodies described in Groups 1A and 1B (14) are similar tothose described in postviral human myocarditis (17), rheu­matic carditis (19), postpericardiotomy and postinfarctionsyndromes (20), adriamycin cardiotoxicity (21) and Chagas'myocarditis (22,23). Studies are currently under way todetermine whether purified heart-specific autoantibodies incoxsackie Bj-infected animals produce myocarditis in un­affected animals or produce damage to cardiac target cells.In addition, Maisch et al. (18) have described the ability ofsera with antisarcolemmal antibody staining patterns to lyserat cardiocytes in vitro.

JACC Vol. 9, No.6June 1987:1311-9

HERSKOWITZ ET AL.COXSACKIEVIRUS B, MURINE MYOCARDITIS

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Conclusions. Several points can be concluded from ourcurrent understanding of murine myocarditis: I) The his­topathologic picture of coxsackievirus B3 myocarditis changesover the course of the illness. 2) Susceptibility to both earlyand late phase myocarditis differs markedly between strainsand is likely a reflection of genetic control (12). The pos­sibility that human myocarditis is equally as complex anentity was recently suggested by other authors (24,25). Thisstudy using multiple inbred mouse strains provides insightsinto the pathogenesis of murine myocarditis and forms abasis for understanding the genetic control of both early andlate phase myocarditis.

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