Congenital Disease Linked to Maternal against Cardiac Myosin Charles R. Cole, Katherine E. Yutzey, Anoop K. Brar, Lisa S. Goessling, Sarah J. VanVickle-Chavez, Madeleine W. This information is current as Cunningham and Pirooz Eghtesady of September 29, 2021. J Immunol published online 26 March 2014 http://www.jimmunol.org/content/early/2014/03/26/jimmun ol.1301264 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 26, 2014, doi:10.4049/jimmunol.1301264 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

Structural congenital heart disease (CHD) has not previously been linked to autoimmunity. In our study, we developed an auto- immune model of structural CHD that resembles hypoplastic left heart syndrome (HLHS), a life-threatening CHD primarily af- fecting 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 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 Downloaded from 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: 000–000.

ongenital heart disease (CHD) is the most common cause heart block, maternal autoantibodies in patients with systemic http://www.jimmunol.org/ of infant death resulting from birth defects (1). Hypo- lupus erythematosus cause injury to the conduction system of the C plastic left heart syndrome (HLHS), a severe and dev- fetal heart (14–16). We had previously hypothesized that auto- astating congenital heart malformation, accounts for nearly 25% might play a role in a maternal–fetal model of of all neonatal deaths from CHD (1–3). HLHS is uniformly fatal structural left-sided CHD (12). Our hypothesis has been sup- without intervention, and despite aggressive medical and surgical ported by the observation of high titers of anti–human cardiac palliation, many affected children experience a significant devel- myosin (CM) IgG autoantibodies in sera from mothers of babies opmental delay and decreased quality of life (4, 5). Although with HLHS, but not other CHD or healthy controls, in an on- etiological mechanisms leading to HLHS are largely unknown, going clinical study (Clinicaltrial.gov 201102410). Anti-CM both genetic and environmental insults are potential contributors autoantibodies are linked to several autoimmune diseases of the by guest on September 29, 2021 (6–10). About one fourth of HLHS cases occur in the context of heart, including autoimmune (17–22) and rheumatic recognized genetic disorders or syndromes; studies involving , the most serious manifestation of group A streptococcus– nonsyndromic family members suggest that heritability is com- induced (23–25). In this study we determined plex (9, 11) and environmental influences such as infection and whether maternal immunization with CM, a major autoantigen in autoimmunity might contribute to the phenotypic expression of human heart (22), could produce an HLHS-like phenotype in certain subsets of HLHS (3, 6, 12, 13). susceptible offspring following transplacental passage of anti- In some cases of CHD, transplacental passage of maternal IgG heart Abs. Experiments conducted in the Lewis rat, an estab- has been reported to affect the fetus. For instance, in congenital lished model of CM-induced autoimmune heart disease (19, 20), led to an HLHS-like phenotype seen in human infants. Autoim- munity against the heart is a new concept in the pathogenesis of *Division of Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Med- HLHS. ical 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, Materials and Methods OK 73104 Ag preparation 1M.W.C. and P.E. are cosenior authors. Rat CM was purified from rat heart tissue according to previously described Received for publication May 22, 2013. Accepted for publication February 18, 2014. techniques, with slight modifications (25, 26). Heart tissue was homoge- This work was supported in part by National Institutes of Health Grants R21- nized in a low-salt buffer [40 mM KCl, 20 mM imidazole, 5 mM ethylene HL104391 (to P.E.), F32- HL103054 (to C.R.C.), R01-HL56267 (to M.W.C.), and glycol-bis(b-aminoethyl ester)-N,N,N’,N’-tetraacetic acid, 5 mM DTT, 0.5 R37-HL35280 (to M.W.C.) and funds from the Saving Tiny Society. M.W.C. mM PMSF, 1 mg leupeptin per milliliter] for 15 s on ice. The washed is the recipient of a National Heart, Lung, and Blood Institute Method to Extend myofibrils were collected by centrifugation at 16,000 3 g for 10 min. The Research in Time Award. pellets were then resuspended in high-salt buffer [0.3 M KCl, 0.15 M Address correspondence and reprint requests to Dr. Pirooz Eghtesady, Pediatric Car- K2HPO4, 1 mM ethylene glycol-bis(b-aminoethyl ester)-N,N,N’,N’-tetra- diothoracic Surgery, Washington University School of Medicine, Campus Box 8234, acetic acid, 5 mM DTT, 0.5 mM PMSF, 1 mg leupeptin per milliliter] and St. Louis, MO 63110. E-mail address: [email protected] homogenized for three 30-s bursts on ice. The homogenized tissue was The online version of this article contains supplemental material. further incubated on ice, with stirring for 30 min to facilitate actomyosin Abbreviations used in this article: b-AR, b-adrenergic receptor; CHD, congenital extraction. After clarification by centrifugation, actomyosin was precipi- heart disease; CM, cardiac myosin; HLHS, hypoplastic left heart syndrome; LV, left tated by the addition of 10 volumes of cold water, followed by a pH ad- ventricle (ventricular); PKA, protein kinase A; RV, right ventricle (ventricular). justment 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 Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 centrifugation at 16,000 3 g. The actomyosin pellet was then resuspended

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301264 2 CONGENITAL HEART DISEASE AND MATERNAL AUTOIMMUNITY in high-salt buffer, ammonium sulfate was increased to 33%, and the KCl ventricular (RV) lateral and apical free walls, three measurements were concentration was increased to 0.5 M. After the actomyosin pellet and salts taken in 100-mm intervals. Three area measurements of the LV and RV were dissolved, ATP was added to 10 mM and MgCl2 was added to 5 mM, were also obtained using comparable apical four-chambered sections. and then the solution was centrifuged at 20,000 3 g for 15 min to remove Measurements were then averaged from each location for statistical actin filaments. The supernatant was removed and stored at 4˚C in the analysis. Owing to some cardiac damage during harvest that altered heart presence of the following inhibitors: 0.5 mM PMSF, 5 mg/ml N-tosyl-L- chamber dimensions, two affected and two unaffected fetal hearts were lysine chloromethyl ketone, and 1 mg leupeptin per milliliter. The presence excluded from LV/RV lumen area measurements. Maternal hearts were of CM was verified and quantitated by ELISA and Western immunoblot processed as previously described (20). A veterinary pathologist evaluated using mAb specific for CM protein. maternal heart sections for the presence of myocarditis and valvulitis. Immunization protocol Western blot analysis Specific pathogen–free female Lewis rats (LEW-RT1l)(∼8 wk old) were Binding of maternal and fetal serum to lysates (10 mg total protein) of adult purchased from Charles River Laboratories (Raleigh, NC) and were rat heart, liver, lung, and spleen was determined by Western blot analysis, maintained in a pathogen-free environment at Cincinnati Children’s Hos- as previously described (22). Sera from CM-injected maternal rats and pital animal facility. Rats were acclimated for 7 d prior to entering the affected fetal offspring, along with sera from control maternal rats and immunization protocol. All animals were treated according to institutional fetal offspring, were analyzed at a dilution of 1:1000. Preincubation of the guidelines and Institutional Animal Care and Use Committee–approved sera with porcine CM (20 mg; Sigma-Aldrich) prior to incubation of the protocols. Female rats were immunized with either rat cardiac CM (n =8) blots was performed to determine specificity. or saline (controls; n = 5). A schematic of the immunization protocol is shown in Supplemental Fig. 1. The rats treated with CM were immunized Immunohistochemistry studies on day 0 by s.c. injection of 1 mg rat CM extract emulsified in CFA at a 1:1 The anti-rat IgG staining was performed as previously described, with slight ratio (v/v) in a total volume of 400 ml. On days 14, 28, and 42 after the modifications, (21) and was g-chain specific for rat IgG. Briefly, mounted Downloaded from m initial immunization, all the rats were boosted i.p. with 500 g extract tissues were baked at 60˚C for 20 min and deparaffinized using a 3:1 ratio m emulsified 1:1 with IFA in a total volume of 200 l. Serum titers of CM of Hemo-D (Fisher) to xylene. After rehydration in graded ethanol washes, Abs were determined by ELISA assays every 7–14 d. Rats with no re- , , tissues were washed twice with PBS, blocked with Protein Block (Bio- sponse ( 1:100) exited the protocol, and rats with medium titers ( 1: Genex, San Ramon, CA) for 30 min at room temperature, and washed 6400) were given up to a total of three additional boosters. In the control twice with PBS. Isotype control goat IgG biotin or biotin-conjugated goat group, CM extract was exchanged for saline in the presence of adjuvant. anti-rat IgG Abs (diluted 1:500; Jackson ImmunoResearch, West Grove, Control animals all received three booster injections. Breeding began 7 d PA) were incubated on tissues overnight at 4˚C in a humidity chamber, after the final booster. No boosters were administered during gestation, http://www.jimmunol.org/ 6 followed by three washes in PBS. Alkaline phosphatase–conjugated which in the Lewis rat is 22 0.2 d. Dams were left with males for 1–3 d, streptavidin was incubated with the tissues at 1 mg/ml for 30 min at room and successful mating was confirmed by the presence of spermatozoa on temperature. After three washes in PBS, Ab binding was detected with Fast a vaginal smear. Vaginal smears were performed daily. Near-term (esti- Red substrate (BioGenex) against a counterstain of Mayer’s hematoxylin mated day of gestation 20 6 1) cesarean section was performed with the (BioGenex). Stained slides were mounted with crystal mount (Fisher), rats under anesthesia (1.5% isoflurane) to deliver the progeny. Fetal ani- dried, and coverslipped using Permount (Sigma Chemical) The amount of mals were harvested following decapitation, and maternal animals after IgG bound is indicated by scoring the intensity of visual Fast Red staining exsanguinations. Maternal and fetal blood was collected during harvest. in the heart tissue. Similar to the previously described process, sections Blood samples were centrifuged at 1300 3 g for 15 min in a fixed angle 2 were deparaffinized and rehydrated and then had antigenic sites unmasked rotor. Serum was collected and stored at 20˚C. Maternal and fetal hearts using the citrate-based solution (#H-3300; Sigma-Aldrich) and high tem- were immediately washed in PBS, fixed in 4% paraformaldehyde, paraffin

perature–pressure protocol of Vector Laboratories. Sections were blocked by guest on September 29, 2021 embedded, sectioned at 7-mm intervals, and histologically analyzed. for 1 h at room temperature with 8% normal goat serum (#G9023; Sigma- Ab quantification by direct ELISA Aldrich), then incubated overnight at 40˚C with primary Abs PHH3 (#06- 570 [1:350]; Millipore), to identify mitotic cells, and MF20 (#MF 20 The assays were conducted as described in previous publications (21, 22, [1:200]; Developmental Studies Hybridoma Bank), to identify myocytes. 25). Immulon 4 (Dynatech) microtiter plates were coated at 4˚C overnight For immunofluorescent detection, sections were incubated with Alexa with rat CM at 10 mg/ml in 0.1 mol/l carbonate–bicarbonate coating buffer Fluor–conjugated secondary Abs (goat anti-rabbit IgG #A11011 and goat (pH 9.6). Plates were washed with PBS containing 0.05% Tween 20 and anti-mouse IgG #A11001 [1:100]; Invitrogen) for 1 h at room temperature, blocked for 1 h at 37˚C with 1% BSA (Fisher Scientific, Hanover Park, IL). followed by a 30-min incubation with the nuclear stain TO-PRO-3 Plates were washed once again with PBS/Tween 20. To determine the rat (#T3605 [1:1000]; Invitrogen). Samples were imaged using a Zeiss LSM anti-CM ELISA Ab titer, rat sera were titrated at an initial dilution of 1:500 510 confocal microscope. The total number of nuclei, the number of nuclei in 1% BSA in PBS buffer and thereafter diluted 2-fold, up to a final di- in myocytes, the total number of pHH3-positive nuclei, and the number of lution of 1:12,800. A total of 50 ml diluted serum was loaded into mi- pHH3-positive nuclei in myocytes were counted for each image, using crotiter wells in duplicates and incubated overnight at 4˚C. Plates were ImageJ software. The total proliferative rate was calculated using total washed 53 with PBS/Tween 20, and 50 ml goat anti-rat IgG (Sigma- pHH3-positive nuclei divided by total nuclei. To determine the mitotic Aldrich, St Louis, MO) conjugated with alkaline phosphatase (1/500 di- index, counts from four areas of the LV, using comparable areas from each lution) was added and incubated at 37˚C for 1 h. Plates were washed with heart, were totaled and used to calculate the percentage of pHH3-positive PBS/Tween 20, and 50 ml substrate para-nitrophenyl phosphate (Sigma- nuclei. TUNEL staining (cat. no. 11 684 809 910; Roche Applied Science) Aldrich) in 0.1 M diethanolamine buffer (pH 9.8) was added to each well. was performed according to the manufacturer’s instruction. Cardiomyocytes OD was measured at 405 nm in an ELISA plate reader (ELx800, BioTek were identified using MF-20 staining, and the cardiomyocyte-specific prolif- Instruments, Winooski, VT). Titers were determined at the highest dilution erative rate was calculated using cardiomyocyte pHH3-positive nuclei divided with an OD value of 0.10 at ∼60 min. Anti-rat CM Ab titers in the ELISA by total cardiomyocyte nuclei. were standardized and controlled using negative and positive control standard sera, obtained from previous experiments. b-adrenergic receptors Statistical analysis b b b m ( -AR) 1 ( 1-AR) and 2 ( 2-AR) (PerkinElmer) were coated at 10 g/ml All statistical analyses were completed using SAS version 9.2. Two vari- onto microtiter plates for testing for rat IgG Abs against the anti–b -AR 1 ables were analyzed using two-sided, independent sample t tests, and three and anti–b -AR Abs in the serum. ELISA was performed according to the 2 variables were analyzed with two-way ANOVA with Tukey–Kramer ad- same procedure stated above. Activation of serum protein kinase A (PKA) justment for multiple comparisons. by the b-AR was performed as previously described (21, 22). Histology and morphometry studies Results Sections from each fetal heart were stained with Movat’s pentachrome Heart defects correlated with elevated anti-CM titers or Masson’s trichrome (both from American Mastertech Scientific). All Female Lewis rats immunized with CM developed peak anti-CM morphometric measurements were obtained using ImageJ software. Com- . parable apical four-chambered sections from each fetal heart were titers ranging from 1:6000 to 1:12,800, prior to photographed (Nikon DS Ri1) and coded to eliminate bias. Two blinded pregnancy (Fig. 1A). Most importantly, all maternal rats (n =8 observers obtained measurements. At the left ventricular (LV) and right mothers with 47 fetuses) with elevated anti-CM Ab titers had at The Journal of Immunology 3

undetectable (,100) anti-CM Ab titers (not shown). Western blot analyses showed specific binding of IgG autoantibodies in mater- nal 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 CM- immunized maternal rat and her affected fetus, but not in con- trol maternal or fetal sera, was blocked by preincubation of the serum with CM. The HLHS-like pathology observed in fetuses from CM-im- munized 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 Downloaded from 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 FIGURE 1. Immunization with CM induces elevated anti-CM Ab titers free wall myocardium displays normal compact myocardium in adult rats and their fetal offspring. (A) Serum titers measured in indi- (Fig. 3E). The severely malformed hearts also had a “spongy” LV http://www.jimmunol.org/ vidual female Lewis rats (8 wk old) immunized with purified rat CM (n =8 myocardium (Fig. 3F). Examination of H&E-stained maternal mothers with 47 fetuses), followed by three to four booster injections heart sections from all CM-immunized adult maternal test rats administered at 2-wk intervals, are shown. Adjuvant was injected in con- trol rats (data not shown). (B) Average fetal (harvested at estimated ges- and adult maternal control rats receiving only adjuvant appeared tational day 20) anti-CM Ab titers in litters of individual adult animals normal and showed no evidence of myocarditis or any abnormal with positive anti-CM Ab response (n = 5 mothers) prior to mating, during histological features in either maternal group. In addition, in the pregnancy, and up to time of harvest. Bars shown are the mean 6 SEM. fetal hearts, no myocarditis or cellular infiltration of the myocar- dium was found. least one offspring with left-sided structural CHD, as determined by histological and morphological analyses. Fetal sera from off- LV primarily affected in rat model with HLHS phenotype by guest on September 29, 2021 spring of CM-immunized mothers had elevated anti-CM Ab titers HLHS is characterized by a reduced, or hypoplastic, LV cavity that that ranged from .1:100 to 1:800 (Fig. 1B). Fetal CM titers is unable to support the systemic circulation, although anatomic of $1:200 correlated with maternal peak CM titers of $1:6400 variation within the classification of HLHS yields a continuum of and/or maternal harvest CM titers of $1:800, confirming positive phenotypic heterogeneity (27, 28). To evaluate chamber-specific transplacental transfer of maternal anti-CM autoantibodies to their structural differences, comparable apical four-chamber sections of progeny. Serum titers of anti-CM Abs in individual fetuses from each heart were studied for cardiac morphometric measurements each litter are shown in Supplemental Fig. 2. The highest anti-CM of the total CM treatment group (n = 47) compared with the ad- Ab titers were observed in maternal rat CM8, who also produced juvant control group (n = 19). RV myocardial thickness of the total the largest number of progeny with structural congenital cardiac CM treatment group was not significantly different from that in malformations (six of nine) (Table I). Control animals, which the adjuvant control group. In contrast, the total CM treatment were injected with adjuvant, and their offspring (n = 19), had 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

Moderately Severely Increased Increased LV Progeny with LV Cavity Loss of Normal LV Myocardial Wall Myocardial Wall Litter Total Maternal Heart Defects, % Hypoplasia, % Valve Structure, % Thickness, % Thickness, % Fetal Litter Size Ab Burden (No. Affected) (No. Affected) (No. Affected) (No. Affected) (No. Affected) CM4 11 3.2 3 105 36 (4) 27 (3) 36 (4) 9 (1) 18 (2) CM5 12 4.0 3 105 8 (1) 8 (1) 8 (1) 8 (1) 0 CM7 8 2.6 3 105 25 (2) 25 (2) 25 (2) 25 (2) 0 CM8 9 7.3 3 105 67 (6) 55 (5) 44 (4) 55 (5) 11 (1) CM10 7 2.3 3 105 29 (2) 29 (2) 14 (1) 29 (2) 0

C1 13 ,100 0 (0) 0 0 0 0 C2 3 ,100 0 (0) 0 0 0 0 C3 3 ,100 0 (0) 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. 4 CONGENITAL HEART DISEASE AND MATERNAL AUTOIMMUNITY Downloaded from

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-im- munized 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). http://www.jimmunol.org/ lateral free wall (p , 0.001) and LV apical free wall (p , 0.05), increased IgG deposition, compared with unaffected fetal hearts compared with the adjuvant control group (Fig. 4A). Statistical (p = 0.03) and adjuvant controls (p = 0.002) (Fig. 5). Moreover, analysis of the affected fetal hearts with the HLHS phenotype IgG deposition in the hearts of offspring of CM-immunized (n = 15) versus the unaffected fetal hearts (n = 32) and adjuvant mothers correlated with the observed cardiac malformations. controls (Fig. 4B) demonstrated no difference in RV myocardial The IgG deposition was principally found in the fetal myocar- thickness between the three groups. Further, the HLHS-like phe- dium, with minimal staining on valve structures or atrioventricular notype in fetal hearts had increased LV lateral free wall thickness, cushions. There was no IgG deposition in maternal heart sections by guest on September 29, 2021 compared with the unaffected fetal hearts (p , 0.0001) and ad- from CM-treated or control groups. juvant controls (p , 0.0001). Affected fetal hearts with the HLHS-like phenotype also demonstrated increased LV apical free Cardiomyocyte proliferation increased in the HLHS phenotype wall thickness, compared with the unaffected fetal hearts (p = 0.0009) Although the causes of certain subtypes of HLHS may originate and adjuvant controls (p = 0.0001). No significant difference was through primary valve defects (7, 28, 33), there is evidence that observed in the LV wall thickness of the unaffected compared with HLHS may result as a consequence of abnormal myocyte pro- adjuvant control fetal hearts. These findings indicate that the ma- liferation during development (34, 35). Further, to determine ternal immune response against CM was associated with increased whether increased cardiomyocyte proliferation contributed to the LV, but not RV, myocardial wall thickness. thickening of the LV myocardium in our model, both compact The significant impact of HLHS results from altered develop- and trabeculated myocardium were examined, as the pathological ment of the LV and left-sided valve structures, characterized by specimens had increased thickness of both. Affected fetal hearts a reduced, or hypoplastic, LV cavity, rendering the heart unable to with the HLHS phenotype had an increased total LV proliferative support the systemic circulation (29, 30). To determine ventricular rate, compared with the unaffected fetal hearts (p = 0.05) and chamber size in our model, the LV lumen area and RV lumen area adjuvant controls (p = 0.003) (Fig. 6A, 6B). The affected fetal of each specimen were measured, and an LV/RV lumen area ratio hearts with the HLHS phenotype had an increased cardiomyocyte- was used to determine relative LV chamber size. The RV served as specific proliferative rate, compared with the unaffected fetal hearts an internal control for this comparison. The affected HLHS-like (p = 0.05) and adjuvant controls (p = 0.002) (Fig. 6C). In contrast, phenotype had a significantly decreased LV/RV lumen area ratio no significant differences were found between groups in the pro- when compared with the unaffected normal fetal hearts (p = 0.002) liferative rate of nonmyocyte nuclei or in apoptosis of the myo- and adjuvant controls (p = 0.007) (Fig. 4C). No significant dif- cardium or valve structures (data not shown). Further, maternal ference in the LV/RV lumen area ratio was noted between unaf- hearts from CM-treated groups did not display any abnormal his- fected and adjuvant control fetal hearts. These findings indicate topathological features or changes in cardiomyocyte proliferation. that the affected fetal hearts had a hypoplastic LV cavity remi- b niscent of HLHS in human infants. Increased anti– -AR titers in affected fetuses Our previous work has shown that anti-CM Abs cross-react with Increased Ab binding in affected hearts the b-AR on the cardiomyocyte surface and induce cAMP-depen- Because maternally acquired IgG is essential in newborn immunity, dent PKA activity in heart cells (21). Because the b-AR also and maternally transferred Abs can mediate tissue injury (31, 32), plays a regulatory role in cardiomyocyte proliferation in early life we examined IgG binding to the fetal hearts in our study. The (36), we measured anti–b-AR titers in affected fetuses that had ele- myocardium of affected fetal hearts with the HLHS phenotype had vated anti-CM titers. We found increased anti–b1-AR (p = 0.007) The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

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 gesta- tional day 20. The RV free wall demonstrates dimensions similar to those of control. The arrow indicates the mitral valve (MV). The brackets depict by guest on September 29, 2021 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” myocar- dium. (A and B) Original magnification 320; scale bar, 600 mm. (C and D) Original magnification 3200; scale bar, 60 mm. (E and F) Original mag- FIGURE 4. Increased LV myocardial thickness and LV cavity hypo- nification 3400; scale bar, 30 mm. All sections (A–F) were stained with plasia are present in affected hearts from CM-immunized mothers. (A) The Movat’s pentachrome. CM-immunized group (n = 47 fetuses) had increased lateral free wall thickness (**p = 0.0009) and increased LVapical free wall thickness (*p = 0.02), compared with adjuvant controls (n = 19 fetuses). There was no (Fig. 7A) and anti–b2-AR (p , 0.0001) (Fig. 7B) Ab titers in fetal difference in RV thickness between groups. (B) Affected fetal hearts (n = sera from the CM treatment group, compared with adjuvant 15) had increased LV lateral free wall thickness compared with unaffected control sera. Because b-ARs on the heart cell surface stimulate fetal hearts (n = 32) (**p = ,0.0001) and adjuvant controls (n = 19) (**p = cAMP-dependent PKA activity, we next incubated fetal sera with ,0.0001). Affected fetal hearts had increased LV apical free wall thick- cultured rat heart cells (H9c2 primary cells) to determine if sera ness, compared with unaffected fetal hearts (**p = 0.0009) and adjuvant controls (**p = 0.0001). There was no difference in RV lateral or apical from CM-treated animals could modulate PKA activity. We found myocardial thickness between groups. (C) Affected fetal hearts (n = 13) a significant increase in PKA activity above basal levels only in had decreased LV/RV lumen area ratio compared with unaffected fetal fetal sera from the CM treatment group that developed heart hearts (n = 30) (**p = 0.002) and adjuvant controls (n = 19) (**p = 0.007), disease (affected group) compared with fetuses from the CM where ** refers to comparisons of the affected with both unaffected and treatment group that were unaffected (p = 0.00023) or controls control groups, indicating that affected hearts had reduced, or hypoplastic, (p = 0.0005) (Fig. 8). No significant difference was noted in PKA LV cavity dimensions. Two variables (A) were analyzed using the two- activity in sera from unaffected fetuses from the CM group or sided independent t test. Three variables (B, C) were analyzed using controls (p = 0.1849). two-way ANOVA with Tukey–Kramer adjustment for multiple compar- isons. Bars shown are the mean 6 SEM. All bars represent the average of RV and LV dimensions for morphometric analysis of each group. Discussion This study presents data supporting a novel concept that defines an autoantibodies against CM, including heart-specific binding of HLHS-like phenotype caused by a maternal autoimmune response CM-immunized maternal and affected fetal serum, IgG deposition against CM. Observations in our fetal rat model of elevated in fetal rat hearts, and the appearance of an HLHS-like phenotype, 6 CONGENITAL HEART DISEASE AND MATERNAL AUTOIMMUNITY Downloaded from http://www.jimmunol.org/ 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 pHH3- positive nuclei. (C) Histogram demonstrating that the percentage of pHH3- positive 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 FIGURE 5. Increased IgG deposition in the myocardium of affected fetuses) (**p = 0.003) groups. Myocytes were identified by MF-20 stain hearts. Both affected and unaffected hearts were observed in the CM-im- and manually counted. Myocyte-specific percentage of pHH3-positive by guest on September 29, 2021 munized group (32% of fetal rats developed an HLHS-like phenotype). cells was greater in the affected group than in the unaffected (*p = 0.05) Anti-rat IgG, alkaline phosphatase conjugated, was used to detect IgG, as and adjuvant control (**p = 0.002) groups. Both affected and unaffected indicated by Fast Red substrate against a counterstain of Mayer’s hema- groups were immunized with CM. There is no difference between the toxylin. (A and C) Adjuvant control hearts demonstrated minimal anti-rat unaffected group and the adjuvant control group in either total proliferation IgG staining. (B and D) Affected hearts demonstrated extensive anti-rat or cardiomyocyte-specific proliferation. (A and B) Original magnification IgG binding, indicated by increased Fast Red substrate staining. Left-sided 3400; scale bar, 50 mm. Three variables (C) were analyzed using two-way mitral valve (MV) identified by arrows on the affected heart. There is more ANOVA with Tukey–Kramer adjustment for multiple comparisons. The IgG deposited within the myocardium of the affected hearts compared with data shown are the mean 6 SEM. 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 was increased in the hearts of affected animals, which could deposition, compared with the unaffected group (n = 32 fetuses) (*p = contribute to the reduction of LV cavity size, as in HLHS. The A B 0.03) and adjuvant control (n = 19 fetuses) (**p = 0.002) groups. ( and ) severely malformed rat hearts also displayed a “spongy” LV 3 C D 3 Original 20; scale bar, 600 mm. ( and ) Original magnification 100; myocardium, which has also has been described in histopathology scale bar, 125 mm. Three variables (E) were analyzed using two-way reports of HLHS (38). ANOVA with Tukey–Kramer adjustment for multiple comparisons. Aver- ages of data shown in bars are mean 6 SEM. 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 auto- support the hypothesis of an immune-mediated pathogenic mech- immune animal models, including experimental models of neo- anism in the development of congenital HLHS-like lesions in the natal lupus, in which congenital heart block phenotype was ob- fetal rat heart. served in 20–30% of immunized pups (39). Although it is not Of human cases of HLHS, #70% show a reduced LV cavity clear why the disease process is primarily localized to left-sided surrounded by a thickened LV myocardium (4, 28). Affected fetal heart structures, it is well known that in fetal circulation oxygen- rat hearts from CM-immunized mothers displayed an HLHS and Ab-rich blood returning from the placenta will preferentially phenotype similar to that of human infants, including the char- pass through the foramen ovale into the left side of the heart. acteristic hypoplastic, or decreased, LV cavity dimensions, al- Thus, fetal left-sided heart structures that are exposed to higher though the RV dimensions were preserved. The affected fetal maternal Ab concentrations could be more susceptible to damage hearts also had an increased LV myocardial thickness, loss of than right-sided structures. Further, maternal hearts in animals normal structure of the mitral and aortic valves, and a disorga- immunized against CM did not demonstrate any structural or in- nized myocardium, as seen in HLHS on histopathological as- flammatory cardiac defects of the myocardium or valves, when sessment (3, 35, 37, 38). Moreover, cardiomyocyte proliferation compared with adjuvant injected controls. Thus, our observations The Journal of Immunology 7

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, Downloaded from unaffected versus affected fetuses (*p = 0.00023) and control versus af- fected fetuses (**p = 0.0005), respectively, resulted in significant p values indicated by * and ** above the unaffected and affected bars. No signif- icant difference was noted in sera from unaffected fetuses compared to the control group (p = 0.1849). Two variables analyzed by two-sided inde- pendent t test. Averaged data shown in bars are mean 6 SEM. http://www.jimmunol.org/ 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 FIGURE 7. Increased anti–b-AR Abs and PKA activity in fetal serum of from an in utero insult to the fetus after the completion of primary CM-immunized rats with the HLHS phenotype. Fetal serum from the CM- cardiac morphogenesis (i.e., after the first 8 wk of human preg- immunized group (n = 29) had significantly increased (A) anti–b1-AR Abs nancy) and corresponding to about gestation day 15.5 in the rat (**p = 0.007) and (B) anti–b2-AR Abs (***p , 0.0001), compared with adjuvant controls (n = 15). (34, 37, 43). IgG Ab distribution in our model suggests that the heart defects in affected animals were primarily myocardial in or- suggest that the developing fetal heart is more or uniquely suscep- igin and that the valve abnormalities may be secondary in nature. by guest on September 29, 2021 tible to immune-mediated injury than is the mature adult heart, and We examined a possible mechanism by which the observed anti- that immune responses against CM led to malformations of the LV. CM IgG response in the CM group could lead to the fetal heart The amount of IgG deposition in the myocardium of adult disease in our model. Passive transfer of cross-reactive anti-CM/ rodents immunized with CM has been shown to correlate with anti–b-AR IgG autoantibodies into adult rats can cause myocar- autoimmune manifestations (17, 21). We found that the maternal dial injury (21). Further, immune absorption of circulating auto- immune response against CM was associated with IgG deposition improves cardiac function of patients with cardiomy- coincident with left-sided congenital heart malformations in their opathy (44, 45). In animal models, Ab-induced cardiomyopathy progeny. The relative lack of Ab staining in the valves suggested induced by stimulation of the b1-AR agonist can be prevented by that in our model valvular abnormalities occurred secondary to the pharmacological neutralization of functionally active anti–b1-AR initial myocardial insult. Elevated anti-CM autoantibody titers in Abs or by the elimination of Abs by anti–b1-AR–selective im- both maternal and fetal serum of the CM-immunized group indi- mune absorption (46). Moreover, blocking the b-AR inhibits cated positive transplacental transfer of maternal anti-CM auto- cardiomyocyte proliferation (36), suggesting a key role for the antibodies. Transplacental Ab-mediated injury to the fetal heart is b-AR in the heart. In addition, studies in rats and humans have the proposed mechanism for a variety of diseases of the fetus and shown that removal of IgG or of specific anti-CM and anti-b-AR newborn, including erythroblastosis fetalis (or hemolytic disease of Abs from the sera depletes the PKA activation properties of the the newborn), hypothyroidism, lupus erythematosus, pemphigus sera (21, 22). vulgaris, and thyrotoxicosis (40). There is also precedence for such Studies in our rat model showed that fetal sera contained elevated a mechanism leading to fetal heart disease, in congenital heart IgG autoantibody titers against CM as well as the b-AR, and block. Cardiac injury in congenital heart block is presumed to arise furthermore, only sera from affected fetuses stimulated PKA ac- from the active transplacental transport of maternal IgG Abs into tivity in rat heart cells in culture. These data strengthen our hy- the fetal circulation. In this condition, injury to conduction tissue of pothesis that functional signaling autoantibodies reactive against the fetal heart by autoantibodies leads to destruction of normal both the CM and the b-AR are associated with the observed pacing mechanisms (14–16). HLHS-like phenotype in the Lewis rat. Further, the known cross- Intrauterine and perinatal exposure of the fetus to maternal IgG reactivity between CM and the b-AR may mediate the increased during pregnancy takes place when the IgG is transported from cardiomyocyte proliferation contributing to the thickening of the mother to fetus across the placenta, beginning at ∼12 wk of LV myocardium and, subsequently, to a reduction in LV cavity gestation in humans (14, 32). Early findings of HLHS, as diag- size. Other antigenic targets are also plausible as etiological fac- nosed via prenatal echocardiography, are appreciated between 14 tors in abnormal fetal cardiomyocyte development resulting in an and 24 wk of gestation in nearly all cases (41, 42). This gestational HLHS-like phenotype. Pathogenesis of certain kinds of CHD such period correlates with the chronology of transplacental transfer of as HLHS may be influenced, either wholly or in part, by alterations 8 CONGENITAL HEART DISEASE AND MATERNAL AUTOIMMUNITY in fetal cardiomyocyte proliferation or differentiation caused by 10. Garg, V., A. N. Muth, J. F. Ransom, M. K. Schluterman, R. Barnes, I. N. King, autoantibodies directed to CM or related Ags. P. D. Grossfeld, and D. Srivastava. 2005. Mutations in NOTCH1 cause aortic valve disease. Nature 437: 270–274. In conclusion, our evidence suggests a potential novel autoim- 11. Grossfeld, P. 2007. Hypoplastic left heart syndrome: new insights. Circ. 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