Download Congenital Heart Disease Linked to Maternal Autoimmunity

Congenital Heart Disease Linked to Maternal
Autoimmunity against Cardiac Myosin
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of June 17, 2017.
Charles R. Cole, Katherine E. Yutzey, Anoop K. Brar, Lisa
S. Goessling, Sarah J. VanVickle-Chavez, Madeleine W.
Cunningham and Pirooz Eghtesady
J Immunol 2014; 192:4074-4082; Prepublished online 26
March 2014;
doi: 10.4049/jimmunol.1301264
http://www.jimmunol.org/content/192/9/4074
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Supplementary
Material
The Journal of Immunology
Congenital Heart Disease Linked to Maternal Autoimmunity
against Cardiac Myosin
Charles R. Cole,* Katherine E. Yutzey,* Anoop K. Brar,† Lisa S. Goessling,†
Sarah J. VanVickle-Chavez,† Madeleine W. Cunningham,‡,1 and Pirooz Eghtesady†,1
C
ongenital heart disease (CHD) is the most common cause
of infant death resulting from birth defects (1). Hypoplastic left heart syndrome (HLHS), a severe and devastating congenital heart malformation, accounts for nearly 25%
of all neonatal deaths from CHD (1–3). HLHS is uniformly fatal
without intervention, and despite aggressive medical and surgical
palliation, many affected children experience a significant developmental delay and decreased quality of life (4, 5). Although
etiological mechanisms leading to HLHS are largely unknown,
both genetic and environmental insults are potential contributors
(6–10). About one fourth of HLHS cases occur in the context of
recognized genetic disorders or syndromes; studies involving
nonsyndromic family members suggest that heritability is complex (9, 11) and environmental influences such as infection and
autoimmunity might contribute to the phenotypic expression of
certain subsets of HLHS (3, 6, 12, 13).
In some cases of CHD, transplacental passage of maternal IgG
has been reported to affect the fetus. For instance, in congenital
*Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; †Division of Cardiothoracic Surgery, Washington
University Medical Center, St. Louis, MO 63110; and ‡Department of Microbiology
and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City,
OK 73104
heart block, maternal autoantibodies in patients with systemic
lupus erythematosus cause injury to the conduction system of the
fetal heart (14–16). We had previously hypothesized that autoimmunity might play a role in a maternal–fetal model of
structural left-sided CHD (12). Our hypothesis has been supported by the observation of high titers of anti–human cardiac
myosin (CM) IgG autoantibodies in sera from mothers of babies
with HLHS, but not other CHD or healthy controls, in an ongoing clinical study (Clinicaltrial.gov 201102410). Anti-CM
autoantibodies are linked to several autoimmune diseases of the
heart, including autoimmune myocarditis (17–22) and rheumatic
carditis, the most serious manifestation of group A streptococcus–
induced rheumatic fever (23–25). In this study we determined
whether maternal immunization with CM, a major autoantigen in
human heart (22), could produce an HLHS-like phenotype in
susceptible offspring following transplacental passage of antiheart Abs. Experiments conducted in the Lewis rat, an established model of CM-induced autoimmune heart disease (19, 20),
led to an HLHS-like phenotype seen in human infants. Autoimmunity against the heart is a new concept in the pathogenesis of
HLHS.
Materials and Methods
Ag preparation
1
M.W.C. and P.E. are cosenior authors.
Received for publication May 22, 2013. Accepted for publication February 18, 2014.
This work was supported in part by National Institutes of Health Grants R21HL104391 (to P.E.), F32-HL103054 (to C.R.C.), R01-HL56267 (to M.W.C.), and
R37-HL35280 (to M.W.C.) and funds from the Saving Tiny Hearts Society. M.W.C.
is the recipient of a National Heart, Lung, and Blood Institute Method to Extend
Research in Time Award.
Address correspondence and reprint requests to Dr. Pirooz Eghtesady, Pediatric Cardiothoracic Surgery, Washington University School of Medicine, Campus Box 8234,
St. Louis, MO 63110. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: b-AR, b-adrenergic receptor; CHD, congenital
heart disease; CM, cardiac myosin; HLHS, hypoplastic left heart syndrome; LV, left
ventricle (ventricular); PKA, protein kinase A; RV, right ventricle (ventricular).
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301264
Rat CM was purified from rat heart tissue according to previously described
techniques, with slight modifications (25, 26). Heart tissue was homogenized in a low-salt buffer [40 mM KCl, 20 mM imidazole, 5 mM ethylene
glycol-bis(b-aminoethyl ester)-N,N,N’,N’-tetraacetic acid, 5 mM DTT, 0.5
mM PMSF, 1 mg leupeptin per milliliter] for 15 s on ice. The washed
myofibrils were collected by centrifugation at 16,000 3 g for 10 min. The
pellets were then resuspended in high-salt buffer [0.3 M KCl, 0.15 M
K2HPO4, 1 mM ethylene glycol-bis(b-aminoethyl ester)-N,N,N’,N’-tetraacetic acid, 5 mM DTT, 0.5 mM PMSF, 1 mg leupeptin per milliliter] and
homogenized for three 30-s bursts on ice. The homogenized tissue was
further incubated on ice, with stirring for 30 min to facilitate actomyosin
extraction. After clarification by centrifugation, actomyosin was precipitated by the addition of 10 volumes of cold water, followed by a pH adjustment to 6.5. DTT was added to 5 mM, and the precipitation was
allowed to proceed for 30 min. The actomyosin was then pelleted by
centrifugation at 16,000 3 g. The actomyosin pellet was then resuspended
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Structural congenital heart disease (CHD) has not previously been linked to autoimmunity. In our study, we developed an autoimmune model of structural CHD that resembles hypoplastic left heart syndrome (HLHS), a life-threatening CHD primarily affecting the left ventricle. Because cardiac myosin (CM) is a dominant autoantigen in autoimmune heart disease, we hypothesized
that immunization with CM might lead to transplacental passage of maternal autoantibodies and a prenatal HLHS phenotype in
exposed fetuses. Elevated anti-CM autoantibodies in maternal and fetal sera, as well as IgG reactivity in fetal myocardium, were
correlated with structural CHD that included diminished left ventricular cavity dimensions in the affected progeny. Further, fetuses
that developed a marked HLHS phenotype had elevated serum titers of anti–b-adrenergic receptor Abs, as well as increased
protein kinase A activity, suggesting a potential mechanism for the observed pathological changes. Our maternal–fetal model
presents a new concept linking autoimmunity against CM and cardiomyocyte proliferation with cardinal features of HLHS. To
our knowledge, this report shows the first evidence in support of a novel immune-mediated mechanism for pathogenesis of structural
CHD that may have implications in its future diagnosis and treatment. The Journal of Immunology, 2014, 192: 4074–4082.
The Journal of Immunology
4075
in high-salt buffer, ammonium sulfate was increased to 33%, and the KCl
concentration was increased to 0.5 M. After the actomyosin pellet and salts
were dissolved, ATP was added to 10 mM and MgCl2 was added to 5 mM,
and then the solution was centrifuged at 20,000 3 g for 15 min to remove
actin filaments. The supernatant was removed and stored at 4˚C in the
presence of the following inhibitors: 0.5 mM PMSF, 5 mg/ml N-tosyl-Llysine chloromethyl ketone, and 1 mg leupeptin per milliliter. The presence
of CM was verified and quantitated by ELISA and Western immunoblot
using mAb specific for CM protein.
ventricular (RV) lateral and apical free walls, three measurements were
taken in 100-mm intervals. Three area measurements of the LV and RV
were also obtained using comparable apical four-chambered sections.
Measurements were then averaged from each location for statistical
analysis. Owing to some cardiac damage during harvest that altered heart
chamber dimensions, two affected and two unaffected fetal hearts were
excluded from LV/RV lumen area measurements. Maternal hearts were
processed as previously described (20). A veterinary pathologist evaluated
maternal heart sections for the presence of myocarditis and valvulitis.
Immunization protocol
Western blot analysis
l
Ab quantification by direct ELISA
The assays were conducted as described in previous publications (21, 22,
25). Immulon 4 (Dynatech) microtiter plates were coated at 4˚C overnight
with rat CM at 10 mg/ml in 0.1 mol/l carbonate–bicarbonate coating buffer
(pH 9.6). Plates were washed with PBS containing 0.05% Tween 20 and
blocked for 1 h at 37˚C with 1% BSA (Fisher Scientific, Hanover Park, IL).
Plates were washed once again with PBS/Tween 20. To determine the rat
anti-CM ELISA Ab titer, rat sera were titrated at an initial dilution of 1:500
in 1% BSA in PBS buffer and thereafter diluted 2-fold, up to a final dilution of 1:12,800. A total of 50 ml diluted serum was loaded into microtiter wells in duplicates and incubated overnight at 4˚C. Plates were
washed 53 with PBS/Tween 20, and 50 ml goat anti-rat IgG (SigmaAldrich, St Louis, MO) conjugated with alkaline phosphatase (1/500 dilution) was added and incubated at 37˚C for 1 h. Plates were washed with
PBS/Tween 20, and 50 ml substrate para-nitrophenyl phosphate (SigmaAldrich) in 0.1 M diethanolamine buffer (pH 9.8) was added to each well.
OD was measured at 405 nm in an ELISA plate reader (ELx800, BioTek
Instruments, Winooski, VT). Titers were determined at the highest dilution
with an OD value of 0.10 at ∼60 min. Anti-rat CM Ab titers in the ELISA
were standardized and controlled using negative and positive control
standard sera, obtained from previous experiments. b-adrenergic receptors
(b-AR) 1 (b1-AR) and 2 (b2-AR) (PerkinElmer) were coated at 10 mg/ml
onto microtiter plates for testing for rat IgG Abs against the anti–b1-AR
and anti–b2-AR Abs in the serum. ELISA was performed according to the
same procedure stated above. Activation of serum protein kinase A (PKA)
by the b-AR was performed as previously described (21, 22).
Binding of maternal and fetal serum to lysates (10 mg total protein) of adult
rat heart, liver, lung, and spleen was determined by Western blot analysis,
as previously described (22). Sera from CM-injected maternal rats and
affected fetal offspring, along with sera from control maternal rats and
fetal offspring, were analyzed at a dilution of 1:1000. Preincubation of the
sera with porcine CM (20 mg; Sigma-Aldrich) prior to incubation of the
blots was performed to determine specificity.
Immunohistochemistry studies
The anti-rat IgG staining was performed as previously described, with slight
modifications, (21) and was g-chain specific for rat IgG. Briefly, mounted
tissues were baked at 60˚C for 20 min and deparaffinized using a 3:1 ratio
of Hemo-D (Fisher) to xylene. After rehydration in graded ethanol washes,
tissues were washed twice with PBS, blocked with Protein Block (BioGenex, San Ramon, CA) for 30 min at room temperature, and washed
twice with PBS. Isotype control goat IgG biotin or biotin-conjugated goat
anti-rat IgG Abs (diluted 1:500; Jackson ImmunoResearch, West Grove,
PA) were incubated on tissues overnight at 4˚C in a humidity chamber,
followed by three washes in PBS. Alkaline phosphatase–conjugated
streptavidin was incubated with the tissues at 1 mg/ml for 30 min at room
temperature. After three washes in PBS, Ab binding was detected with Fast
Red substrate (BioGenex) against a counterstain of Mayer’s hematoxylin
(BioGenex). Stained slides were mounted with crystal mount (Fisher),
dried, and coverslipped using Permount (Sigma Chemical) The amount of
IgG bound is indicated by scoring the intensity of visual Fast Red staining
in the heart tissue. Similar to the previously described process, sections
were deparaffinized and rehydrated and then had antigenic sites unmasked
using the citrate-based solution (#H-3300; Sigma-Aldrich) and high temperature–pressure protocol of Vector Laboratories. Sections were blocked
for 1 h at room temperature with 8% normal goat serum (#G9023; SigmaAldrich), then incubated overnight at 40˚C with primary Abs PHH3 (#06570 [1:350]; Millipore), to identify mitotic cells, and MF20 (#MF 20
[1:200]; Developmental Studies Hybridoma Bank), to identify myocytes.
For immunofluorescent detection, sections were incubated with Alexa
Fluor–conjugated secondary Abs (goat anti-rabbit IgG #A11011 and goat
anti-mouse IgG #A11001 [1:100]; Invitrogen) for 1 h at room temperature,
followed by a 30-min incubation with the nuclear stain TO-PRO-3
(#T3605 [1:1000]; Invitrogen). Samples were imaged using a Zeiss LSM
510 confocal microscope. The total number of nuclei, the number of nuclei
in myocytes, the total number of pHH3-positive nuclei, and the number of
pHH3-positive nuclei in myocytes were counted for each image, using
ImageJ software. The total proliferative rate was calculated using total
pHH3-positive nuclei divided by total nuclei. To determine the mitotic
index, counts from four areas of the LV, using comparable areas from each
heart, were totaled and used to calculate the percentage of pHH3-positive
nuclei. TUNEL staining (cat. no. 11 684 809 910; Roche Applied Science)
was performed according to the manufacturer’s instruction. Cardiomyocytes
were identified using MF-20 staining, and the cardiomyocyte-specific proliferative rate was calculated using cardiomyocyte pHH3-positive nuclei divided
by total cardiomyocyte nuclei.
Statistical analysis
All statistical analyses were completed using SAS version 9.2. Two variables were analyzed using two-sided, independent sample t tests, and three
variables were analyzed with two-way ANOVA with Tukey–Kramer adjustment for multiple comparisons.
Histology and morphometry studies
Results
Sections from each fetal heart were stained with Movat’s pentachrome
or Masson’s trichrome (both from American Mastertech Scientific). All
morphometric measurements were obtained using ImageJ software. Comparable apical four-chambered sections from each fetal heart were
photographed (Nikon DS Ri1) and coded to eliminate bias. Two blinded
observers obtained measurements. At the left ventricular (LV) and right
Heart defects correlated with elevated anti-CM titers
Female Lewis rats immunized with CM developed peak anti-CM
autoantibody titers ranging from 1:6000 to .1:12,800, prior to
pregnancy (Fig. 1A). Most importantly, all maternal rats (n = 8
mothers with 47 fetuses) with elevated anti-CM Ab titers had at
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Specific pathogen–free female Lewis rats (LEW-RT1 ) (∼8 wk old) were
purchased from Charles River Laboratories (Raleigh, NC) and were
maintained in a pathogen-free environment at Cincinnati Children’s Hospital animal facility. Rats were acclimated for 7 d prior to entering the
immunization protocol. All animals were treated according to institutional
guidelines and Institutional Animal Care and Use Committee–approved
protocols. Female rats were immunized with either rat cardiac CM (n = 8)
or saline (controls; n = 5). A schematic of the immunization protocol is
shown in Supplemental Fig. 1. The rats treated with CM were immunized
on day 0 by s.c. injection of 1 mg rat CM extract emulsified in CFA at a 1:1
ratio (v/v) in a total volume of 400 ml. On days 14, 28, and 42 after the
initial immunization, all the rats were boosted i.p. with 500 mg extract
emulsified 1:1 with IFA in a total volume of 200 ml. Serum titers of CM
Abs were determined by ELISA assays every 7–14 d. Rats with no response (,1:100) exited the protocol, and rats with medium titers (,1:
6400) were given up to a total of three additional boosters. In the control
group, CM extract was exchanged for saline in the presence of adjuvant.
Control animals all received three booster injections. Breeding began 7 d
after the final booster. No boosters were administered during gestation,
which in the Lewis rat is 22 6 0.2 d. Dams were left with males for 1–3 d,
and successful mating was confirmed by the presence of spermatozoa on
a vaginal smear. Vaginal smears were performed daily. Near-term (estimated day of gestation 20 6 1) cesarean section was performed with the
rats under anesthesia (1.5% isoflurane) to deliver the progeny. Fetal animals were harvested following decapitation, and maternal animals after
exsanguinations. Maternal and fetal blood was collected during harvest.
Blood samples were centrifuged at 1300 3 g for 15 min in a fixed angle
rotor. Serum was collected and stored at 220˚C. Maternal and fetal hearts
were immediately washed in PBS, fixed in 4% paraformaldehyde, paraffin
embedded, sectioned at 7-mm intervals, and histologically analyzed.
4076
CONGENITAL HEART DISEASE AND MATERNAL AUTOIMMUNITY
FIGURE 1. Immunization with CM induces elevated anti-CM Ab titers
in adult rats and their fetal offspring. (A) Serum titers measured in individual female Lewis rats (8 wk old) immunized with purified rat CM (n = 8
mothers with 47 fetuses), followed by three to four booster injections
administered at 2-wk intervals, are shown. Adjuvant was injected in control rats (data not shown). (B) Average fetal (harvested at estimated gestational day 20) anti-CM Ab titers in litters of individual adult animals
with positive anti-CM Ab response (n = 5 mothers) prior to mating, during
pregnancy, and up to time of harvest. Bars shown are the mean 6 SEM.
least one offspring with left-sided structural CHD, as determined
by histological and morphological analyses. Fetal sera from offspring of CM-immunized mothers had elevated anti-CM Ab titers
that ranged from .1:100 to 1:800 (Fig. 1B). Fetal CM titers
of $1:200 correlated with maternal peak CM titers of $1:6400
and/or maternal harvest CM titers of $1:800, confirming positive
transplacental transfer of maternal anti-CM autoantibodies to their
progeny. Serum titers of anti-CM Abs in individual fetuses from
each litter are shown in Supplemental Fig. 2. The highest anti-CM
Ab titers were observed in maternal rat CM8, who also produced
the largest number of progeny with structural congenital cardiac
malformations (six of nine) (Table I). Control animals, which
were injected with adjuvant, and their offspring (n = 19), had
LV primarily affected in rat model with HLHS phenotype
HLHS is characterized by a reduced, or hypoplastic, LV cavity that
is unable to support the systemic circulation, although anatomic
variation within the classification of HLHS yields a continuum of
phenotypic heterogeneity (27, 28). To evaluate chamber-specific
structural differences, comparable apical four-chamber sections of
each heart were studied for cardiac morphometric measurements
of the total CM treatment group (n = 47) compared with the adjuvant control group (n = 19). RV myocardial thickness of the total
CM treatment group was not significantly different from that in
the adjuvant control group. In contrast, the total CM treatment
group had significantly increased myocardial thickness of the LV
Table I. Maternal immune response against CM and associated left-sided structural congenital cardiac abnormalities in the progeny
Fetal Litter
Litter
Size
CM4
CM5
CM7
CM8
CM10
11
12
8
9
7
C1
C2
C3
13
3
3
Total Maternal
Ab Burden
3.2
4.0
2.6
7.3
2.3
3
3
3
3
3
105
105
105
105
105
,100
,100
,100
Progeny with
Heart Defects, %
(No. Affected)
36
8
25
67
29
(4)
(1)
(2)
(6)
(2)
0 (0)
0 (0)
0 (0)
LV Cavity
Hypoplasia, %
(No. Affected)
27
8
25
55
29
(3)
(1)
(2)
(5)
(2)
0
0
0
Loss of Normal
Valve Structure, %
(No. Affected)
36
8
25
44
14
(4)
(1)
(2)
(4)
(1)
0
0
0
Severely Increased
LV Myocardial Wall
Thickness, %
(No. Affected)
9
8
25
55
29
(1)
(1)
(2)
(5)
(2)
0
0
0
Moderately
Increased LV
Myocardial Wall
Thickness, %
(No. Affected)
18 (2)
0
0
11 (1)
0
0
0
0
The total maternal Ab burden was calculated using area under the curve of the maternal Ab titer graph (Fig. 1). The values given are the mean 6 SEM of each liter. Neither
maternal nor fetal adjuvant-injected control animals (C1–3) had elevated anti-CM Ab titers.
C, adjuvant injected controls; CM, CM-immunized; D, day.
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undetectable (,100) anti-CM Ab titers (not shown). Western blot
analyses showed specific binding of IgG autoantibodies in maternal and fetal serum to adult rat cardiac tissue lysates, but not
to rat kidney, lung, or spleen (Fig. 2). Further, binding of this
cardiac-specific band at 200 kDa present in sera of a CMimmunized maternal rat and her affected fetus, but not in control maternal or fetal sera, was blocked by preincubation of the
serum with CM.
The HLHS-like pathology observed in fetuses from CM-immunized mother rats is illustrated in Fig. 3. We observed that
32% (15 of 47) of fetuses in the CM treatment group developed
a left-sided structural CHD and 28% (13 of 47) of the fetuses had
reduced or hypoplastic LV cavities (Fig. 3B, 3D), whereas none of
the control fetuses (n = 19) had cardiac abnormalities (Fig. 3A,
3C). The congenitally malformed, affected fetal hearts with an
HLHS phenotype had a thickened LV myocardium (30%; 14 of
47) and/or loss of normal mitral and aortic valve structure (26%;
12 of 47) (Fig. 3D). The left-sided valve structures displayed loss
of smooth rounded edges and were foreshortened. The affected
fetal hearts that were severely malformed (23%; 11 of 47) showed
a 50–160% increase in LV myocardial wall thickness, whereas the
moderately malformed hearts (6%; 3 of 47) displayed a 15–50%
increase in LV wall thickness. In adjuvant controls, the fetal LV
free wall myocardium displays normal compact myocardium
(Fig. 3E). The severely malformed hearts also had a “spongy” LV
myocardium (Fig. 3F). Examination of H&E-stained maternal
heart sections from all CM-immunized adult maternal test rats
and adult maternal control rats receiving only adjuvant appeared
normal and showed no evidence of myocarditis or any abnormal
histological features in either maternal group. In addition, in the
fetal hearts, no myocarditis or cellular infiltration of the myocardium was found.
The Journal of Immunology
4077
lateral free wall (p , 0.001) and LV apical free wall (p , 0.05),
compared with the adjuvant control group (Fig. 4A). Statistical
analysis of the affected fetal hearts with the HLHS phenotype
(n = 15) versus the unaffected fetal hearts (n = 32) and adjuvant
controls (Fig. 4B) demonstrated no difference in RV myocardial
thickness between the three groups. Further, the HLHS-like phenotype in fetal hearts had increased LV lateral free wall thickness,
compared with the unaffected fetal hearts (p , 0.0001) and adjuvant controls (p , 0.0001). Affected fetal hearts with the
HLHS-like phenotype also demonstrated increased LV apical free
wall thickness, compared with the unaffected fetal hearts (p = 0.0009)
and adjuvant controls (p = 0.0001). No significant difference was
observed in the LV wall thickness of the unaffected compared with
adjuvant control fetal hearts. These findings indicate that the maternal immune response against CM was associated with increased
LV, but not RV, myocardial wall thickness.
The significant impact of HLHS results from altered development of the LV and left-sided valve structures, characterized by
a reduced, or hypoplastic, LV cavity, rendering the heart unable to
support the systemic circulation (29, 30). To determine ventricular
chamber size in our model, the LV lumen area and RV lumen area
of each specimen were measured, and an LV/RV lumen area ratio
was used to determine relative LV chamber size. The RV served as
an internal control for this comparison. The affected HLHS-like
phenotype had a significantly decreased LV/RV lumen area ratio
when compared with the unaffected normal fetal hearts (p = 0.002)
and adjuvant controls (p = 0.007) (Fig. 4C). No significant difference in the LV/RV lumen area ratio was noted between unaffected and adjuvant control fetal hearts. These findings indicate
that the affected fetal hearts had a hypoplastic LV cavity reminiscent of HLHS in human infants.
Increased Ab binding in affected hearts
Because maternally acquired IgG is essential in newborn immunity,
and maternally transferred Abs can mediate tissue injury (31, 32),
we examined IgG binding to the fetal hearts in our study. The
myocardium of affected fetal hearts with the HLHS phenotype had
increased IgG deposition, compared with unaffected fetal hearts
(p = 0.03) and adjuvant controls (p = 0.002) (Fig. 5). Moreover,
IgG deposition in the hearts of offspring of CM-immunized
mothers correlated with the observed cardiac malformations.
The IgG deposition was principally found in the fetal myocardium, with minimal staining on valve structures or atrioventricular
cushions. There was no IgG deposition in maternal heart sections
from CM-treated or control groups.
Cardiomyocyte proliferation increased in the HLHS phenotype
Although the causes of certain subtypes of HLHS may originate
through primary valve defects (7, 28, 33), there is evidence that
HLHS may result as a consequence of abnormal myocyte proliferation during development (34, 35). Further, to determine
whether increased cardiomyocyte proliferation contributed to the
thickening of the LV myocardium in our model, both compact
and trabeculated myocardium were examined, as the pathological
specimens had increased thickness of both. Affected fetal hearts
with the HLHS phenotype had an increased total LV proliferative
rate, compared with the unaffected fetal hearts (p = 0.05) and
adjuvant controls (p = 0.003) (Fig. 6A, 6B). The affected fetal
hearts with the HLHS phenotype had an increased cardiomyocytespecific proliferative rate, compared with the unaffected fetal hearts
(p = 0.05) and adjuvant controls (p = 0.002) (Fig. 6C). In contrast,
no significant differences were found between groups in the proliferative rate of nonmyocyte nuclei or in apoptosis of the myocardium or valve structures (data not shown). Further, maternal
hearts from CM-treated groups did not display any abnormal histopathological features or changes in cardiomyocyte proliferation.
Increased anti–b-AR titers in affected fetuses
Our previous work has shown that anti-CM Abs cross-react with
the b-AR on the cardiomyocyte surface and induce cAMP-dependent PKA activity in heart cells (21). Because the b-AR also
plays a regulatory role in cardiomyocyte proliferation in early life
(36), we measured anti–b-AR titers in affected fetuses that had elevated anti-CM titers. We found increased anti–b1-AR (p = 0.007)
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FIGURE 2. Western blot analysis of serum shows heart-specific binding. Sera from a control maternal rat (C7) and a fetal offspring, and a CM-immunized maternal rat (M8) and an affected fetal offspring (M8F4) were incubated with tissue extracts (10 mg each) of adult heart, liver, and spleen. The
heart-specific binding by sera from the CM-immunized mother and her affected fetus, but not control sera, is blocked by preincubating the sera with CM
(20 mg).
4078
CONGENITAL HEART DISEASE AND MATERNAL AUTOIMMUNITY
(Fig. 7A) and anti–b2-AR (p , 0.0001) (Fig. 7B) Ab titers in fetal
sera from the CM treatment group, compared with adjuvant
control sera. Because b-ARs on the heart cell surface stimulate
cAMP-dependent PKA activity, we next incubated fetal sera with
cultured rat heart cells (H9c2 primary cells) to determine if sera
from CM-treated animals could modulate PKA activity. We found
a significant increase in PKA activity above basal levels only in
fetal sera from the CM treatment group that developed heart
disease (affected group) compared with fetuses from the CM
treatment group that were unaffected (p = 0.00023) or controls
(p = 0.0005) (Fig. 8). No significant difference was noted in PKA
activity in sera from unaffected fetuses from the CM group or
controls (p = 0.1849).
Discussion
This study presents data supporting a novel concept that defines an
HLHS-like phenotype caused by a maternal autoimmune response
against CM. Observations in our fetal rat model of elevated
FIGURE 4. Increased LV myocardial thickness and LV cavity hypoplasia are present in affected hearts from CM-immunized mothers. (A) The
CM-immunized group (n = 47 fetuses) had increased lateral free wall
thickness (**p = 0.0009) and increased LV apical free wall thickness (*p =
0.02), compared with adjuvant controls (n = 19 fetuses). There was no
difference in RV thickness between groups. (B) Affected fetal hearts (n =
15) had increased LV lateral free wall thickness compared with unaffected
fetal hearts (n = 32) (**p = ,0.0001) and adjuvant controls (n = 19) (**p =
,0.0001). Affected fetal hearts had increased LV apical free wall thickness, compared with unaffected fetal hearts (**p = 0.0009) and adjuvant
controls (**p = 0.0001). There was no difference in RV lateral or apical
myocardial thickness between groups. (C) Affected fetal hearts (n = 13)
had decreased LV/RV lumen area ratio compared with unaffected fetal
hearts (n = 30) (**p = 0.002) and adjuvant controls (n = 19) (**p = 0.007),
where ** refers to comparisons of the affected with both unaffected and
control groups, indicating that affected hearts had reduced, or hypoplastic,
LV cavity dimensions. Two variables (A) were analyzed using the twosided independent t test. Three variables (B, C) were analyzed using
two-way ANOVA with Tukey–Kramer adjustment for multiple comparisons. Bars shown are the mean 6 SEM. All bars represent the average of
RV and LV dimensions for morphometric analysis of each group.
autoantibodies against CM, including heart-specific binding of
CM-immunized maternal and affected fetal serum, IgG deposition
in fetal rat hearts, and the appearance of an HLHS-like phenotype,
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FIGURE 3. Affected fetal rat hearts demonstrate left-sided structural
cardiac malformations that are characteristic of HLHS-like CHD. (A)
Adjuvant control fetal rat heart with normal heart structures at estimated
gestational day 20. (B) Affected fetal rat heart demonstrated a hypoplastic
LV cavity with a thickened LV free wall and septum at estimated gestational day 20. The RV free wall demonstrates dimensions similar to those
of control. The arrow indicates the mitral valve (MV). The brackets depict
LV lateral and apical free walls. (C) Representative adjuvant control fetal
heart shows MV with normal structure. (D) MV of affected fetal rat heart
demonstrates loss of normal structure. The valve does not have smooth,
rounded edges and appears foreshortened. (E) Adjuvant control fetal LV
free wall myocardium displays normal compact myocardium. (F) Affected
heart displays myocyte disarray and “spongy” myocardium of LV free
wall. Arrows indicate areas of myocyte disarray with “spongy” myocardium. (A and B) Original magnification 320; scale bar, 600 mm. (C and D)
Original magnification 3200; scale bar, 60 mm. (E and F) Original magnification 3400; scale bar, 30 mm. All sections (A–F) were stained with
Movat’s pentachrome.
The Journal of Immunology
support the hypothesis of an immune-mediated pathogenic mechanism in the development of congenital HLHS-like lesions in the
fetal rat heart.
Of human cases of HLHS, #70% show a reduced LV cavity
surrounded by a thickened LV myocardium (4, 28). Affected fetal
rat hearts from CM-immunized mothers displayed an HLHS
phenotype similar to that of human infants, including the characteristic hypoplastic, or decreased, LV cavity dimensions, although the RV dimensions were preserved. The affected fetal
hearts also had an increased LV myocardial thickness, loss of
normal structure of the mitral and aortic valves, and a disorganized myocardium, as seen in HLHS on histopathological assessment (3, 35, 37, 38). Moreover, cardiomyocyte proliferation
FIGURE 6. Increased total proliferation and cardiomyocyte-specific
proliferation of the LV myocardium in affected hearts with the HLHS
phenotype. (A and B) Adjuvant control and affected hearts stained with
pHH3, MF-20, and TO-PRO-3. There were more pHH3-positive nuclei in
the affected heart compared with adjuvant control. Arrows indicate pHH3positive nuclei. (C) Histogram demonstrating that the percentage of pHH3positive total cells was greater in the affected group (n = 15) compared
with the unaffected group (n = 32) (*p = 0.05) and adjuvant control (n = 19
fetuses) (**p = 0.003) groups. Myocytes were identified by MF-20 stain
and manually counted. Myocyte-specific percentage of pHH3-positive
cells was greater in the affected group than in the unaffected (*p = 0.05)
and adjuvant control (**p = 0.002) groups. Both affected and unaffected
groups were immunized with CM. There is no difference between the
unaffected group and the adjuvant control group in either total proliferation
or cardiomyocyte-specific proliferation. (A and B) Original magnification
3400; scale bar, 50 mm. Three variables (C) were analyzed using two-way
ANOVA with Tukey–Kramer adjustment for multiple comparisons. The
data shown are the mean 6 SEM.
was increased in the hearts of affected animals, which could
contribute to the reduction of LV cavity size, as in HLHS. The
severely malformed rat hearts also displayed a “spongy” LV
myocardium, which has also has been described in histopathology
reports of HLHS (38).
The development of the congenital HLHS-like phenotype in our
model in association with elevated titers against CM occurred in
∼32% of fetal rats. This rate is comparable to that in other autoimmune animal models, including experimental models of neonatal lupus, in which congenital heart block phenotype was observed in 20–30% of immunized pups (39). Although it is not
clear why the disease process is primarily localized to left-sided
heart structures, it is well known that in fetal circulation oxygenand Ab-rich blood returning from the placenta will preferentially
pass through the foramen ovale into the left side of the heart.
Thus, fetal left-sided heart structures that are exposed to higher
maternal Ab concentrations could be more susceptible to damage
than right-sided structures. Further, maternal hearts in animals
immunized against CM did not demonstrate any structural or inflammatory cardiac defects of the myocardium or valves, when
compared with adjuvant injected controls. Thus, our observations
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FIGURE 5. Increased IgG deposition in the myocardium of affected
hearts. Both affected and unaffected hearts were observed in the CM-immunized group (32% of fetal rats developed an HLHS-like phenotype).
Anti-rat IgG, alkaline phosphatase conjugated, was used to detect IgG, as
indicated by Fast Red substrate against a counterstain of Mayer’s hematoxylin. (A and C) Adjuvant control hearts demonstrated minimal anti-rat
IgG staining. (B and D) Affected hearts demonstrated extensive anti-rat
IgG binding, indicated by increased Fast Red substrate staining. Left-sided
mitral valve (MV) identified by arrows on the affected heart. There is more
IgG deposited within the myocardium of the affected hearts compared with
valve structures. PBS-treated control sections did not stain red and were
blue and negative for IgG (not shown). (E) Scored results for amount of
anti-rat IgG deposition. The affected group (n = 15) had increased IgG
deposition, compared with the unaffected group (n = 32 fetuses) (*p =
0.03) and adjuvant control (n = 19 fetuses) (**p = 0.002) groups. (A and B)
Original 320; scale bar, 600 mm. (C and D) Original magnification 3100;
scale bar, 125 mm. Three variables (E) were analyzed using two-way
ANOVA with Tukey–Kramer adjustment for multiple comparisons. Averages of data shown in bars are mean 6 SEM.
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CONGENITAL HEART DISEASE AND MATERNAL AUTOIMMUNITY
FIGURE 7. Increased anti–b-AR Abs and PKA activity in fetal serum of
CM-immunized rats with the HLHS phenotype. Fetal serum from the CMimmunized group (n = 29) had significantly increased (A) anti–b1-AR Abs
(**p = 0.007) and (B) anti–b2-AR Abs (***p , 0.0001), compared with
adjuvant controls (n = 15).
suggest that the developing fetal heart is more or uniquely susceptible to immune-mediated injury than is the mature adult heart, and
that immune responses against CM led to malformations of the LV.
The amount of IgG deposition in the myocardium of adult
rodents immunized with CM has been shown to correlate with
autoimmune manifestations (17, 21). We found that the maternal
immune response against CM was associated with IgG deposition
coincident with left-sided congenital heart malformations in their
progeny. The relative lack of Ab staining in the valves suggested
that in our model valvular abnormalities occurred secondary to the
initial myocardial insult. Elevated anti-CM autoantibody titers in
both maternal and fetal serum of the CM-immunized group indicated positive transplacental transfer of maternal anti-CM autoantibodies. Transplacental Ab-mediated injury to the fetal heart is
the proposed mechanism for a variety of diseases of the fetus and
newborn, including erythroblastosis fetalis (or hemolytic disease of
the newborn), hypothyroidism, lupus erythematosus, pemphigus
vulgaris, and thyrotoxicosis (40). There is also precedence for such
a mechanism leading to fetal heart disease, in congenital heart
block. Cardiac injury in congenital heart block is presumed to arise
from the active transplacental transport of maternal IgG Abs into
the fetal circulation. In this condition, injury to conduction tissue of
the fetal heart by autoantibodies leads to destruction of normal
pacing mechanisms (14–16).
Intrauterine and perinatal exposure of the fetus to maternal IgG
during pregnancy takes place when the IgG is transported from
mother to fetus across the placenta, beginning at ∼12 wk of
gestation in humans (14, 32). Early findings of HLHS, as diagnosed via prenatal echocardiography, are appreciated between 14
and 24 wk of gestation in nearly all cases (41, 42). This gestational
period correlates with the chronology of transplacental transfer of
maternal IgG when maternally transferred Abs can mediate tissue
injury (14). Recent work has demonstrated that myocytes in
HLHS are well differentiated (37), suggesting that HLHS results
from an in utero insult to the fetus after the completion of primary
cardiac morphogenesis (i.e., after the first 8 wk of human pregnancy) and corresponding to about gestation day 15.5 in the rat
(34, 37, 43). IgG Ab distribution in our model suggests that the
heart defects in affected animals were primarily myocardial in origin and that the valve abnormalities may be secondary in nature.
We examined a possible mechanism by which the observed antiCM IgG response in the CM group could lead to the fetal heart
disease in our model. Passive transfer of cross-reactive anti-CM/
anti–b-AR IgG autoantibodies into adult rats can cause myocardial injury (21). Further, immune absorption of circulating autoantibodies improves cardiac function of patients with cardiomyopathy (44, 45). In animal models, Ab-induced cardiomyopathy
induced by stimulation of the b1-AR agonist can be prevented by
pharmacological neutralization of functionally active anti–b1-AR
Abs or by the elimination of Abs by anti–b1-AR–selective immune absorption (46). Moreover, blocking the b-AR inhibits
cardiomyocyte proliferation (36), suggesting a key role for the
b-AR in the heart. In addition, studies in rats and humans have
shown that removal of IgG or of specific anti-CM and anti-b-AR
Abs from the sera depletes the PKA activation properties of the
sera (21, 22).
Studies in our rat model showed that fetal sera contained elevated
IgG autoantibody titers against CM as well as the b-AR, and
furthermore, only sera from affected fetuses stimulated PKA activity in rat heart cells in culture. These data strengthen our hypothesis that functional signaling autoantibodies reactive against
both the CM and the b-AR are associated with the observed
HLHS-like phenotype in the Lewis rat. Further, the known crossreactivity between CM and the b-AR may mediate the increased
cardiomyocyte proliferation contributing to the thickening of the
LV myocardium and, subsequently, to a reduction in LV cavity
size. Other antigenic targets are also plausible as etiological factors in abnormal fetal cardiomyocyte development resulting in an
HLHS-like phenotype. Pathogenesis of certain kinds of CHD such
as HLHS may be influenced, either wholly or in part, by alterations
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FIGURE 8. PKA activity is significantly (**) increased in fetal serum
from affected fetuses, but not from unaffected offspring (*) or controls. Rat
cardiac myocytes (H9c2 primary cell line) incubated with fetal serum show
a significant increase in PKA activity above basal levels only by fetal sera
from the CM treatment group that developed heart disease (**, affected
group) compared with fetuses from the CM treatment group. In summary,
unaffected versus affected fetuses (*p = 0.00023) and control versus affected fetuses (**p = 0.0005), respectively, resulted in significant p values
indicated by * and ** above the unaffected and affected bars. No significant difference was noted in sera from unaffected fetuses compared to the
control group (p = 0.1849). Two variables analyzed by two-sided independent t test. Averaged data shown in bars are mean 6 SEM.
The Journal of Immunology
Acknowledgments
We thank Mitali Basu and Christopher Lam (Cincinnati Children’s Hospital
Medical Center) for technical assistance with the immunization experiments; R. Scott Baker and Danielle Herbert (Cincinnati Children’s Hospital Medical Center) and Heidi Wagner (Washington University Medical
Center) for tissue sectioning and morphological measurements; Adita
Mascaro-Blanco, Kathy Alvarez, and Stanley Kosanke (University of
Oklahoma Health Sciences Center) for excellent technical support
for the immunoassays, phosphohistone, and immunohistochemistry;
and Dennis Hanseman (University of Cincinnati, Cincinnati, OH) for
statistical support.
Disclosures
M.W.C. is Chief Scientific Officer for Moleculera Labs. The other authors
have no financial conflicts of interest.
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