Congenital heart block (CHB) which occurs without structural abnormalities and is detectable in the second trimester is almost universally associated with maternal IgG autoantibodies reactive with the intracellular soluble ribonucleoproteins 48-kd SSB/La, 52-kd SSA/Ro, and 60-kd SSA/Ro (1). Although this remarkable association suggests an inherent pathogenicity of the antibody, only 2% of neonates born to mothers with these candidate autoantibodies have CHB (2). In fetuses exposed to maternal anti-SSA/Ro and anti-SSB/La antibodies, the pathway to clinical effect may be variable; in most fetuses, the effects are kept in check (normal sinus rhythm), while in others they are subclinical (first-degree block) or, in a very few, are fully executed (advanced block with complete fibrosis of the atrioventricular [AV] node and cardiomyopathy secondary to endocardial fibroelastosis). In addition, the recurrence rates of CHB in subsequent pregnancies approach 20%, not 100% (1). Fetal genetic factors may play a contributory role, but are not fully causative, since identical twins are more often discordant than concordant for the disease (for review, see ref.1). These disparities imply that maternal antibodies are a necessary component of the pathway, and that fetal factors are perhaps additive; however, even when acting in concert, these elements are insufficient to cause damage. The rarity of fully executed CHB may be the consequence of a triple hit: maternal antibodies, fetal factors, and the in utero environment.
Based on the immunohistologic findings of fatal CHB and in vitro coculturing of fetal cardiocytes, macrophages, and fibroblasts in our laboratory, we have proposed a pathologic cascade to cardiac injury (3–5). The scenario initiates with exaggerated apoptosis (observed in the septal tissue of all hearts with CHB studied to date). Apoptosis results in surface translocation of the normally sequestered intracellular target autoantigens, which accounts for the accessibility of these autoantigens to circulating maternal autoantibodies. Secretion of profibrosing cytokines, such as transforming growth factor β (TGFβ), by macrophages that have phagocytosed the opsonized apoptotic cardiocytes promotes the transdifferentiation of the cardiac fibroblasts to a scarring phenotype. The histologic hallmark of CHB is fibrosis, which is remarkable, given the prevailing dogma that fetuses heal without permanent scarring. Equally remarkable is that AV nodal replacement occurs rapidly, with bradyarrhythmia reported, in some cases, within 2 weeks after normal sinus rhythm (see ref.6 and Buyon JP: unpublished observations). Accordingly, focus on the fetal cardiac fibroblast should be a priority of translational studies in CHB.
A potential contribution by hypoxia is underscored by the fact that the fetal heart may be exposed to a relative state of chronic hypoxia, especially when compared with adult hearts (which are not affected despite circulating anti-SSA/Ro antibodies), since the coronary artery in adults, but not fetuses, is the first branch drawing fully oxygenated blood. Moreover, cAMP, a key regulator of homeostasis of extracellular matrix in fibroblasts, may be altered during hypoxic conditions (7). It is possible that cAMP is maintained at relatively high levels during normal development of the fetal heart, as was shown indirectly by a recent report indicating significantly higher levels of cAMP in cord blood compared with that obtained from adults (8).
Accordingly, the present study was initiated to experimentally address the effect of hypoxia and TGFβ on the fibrosing component of CHB. Evidence of hypoxic exposure was sought by immunohistologic evaluation of the cardiac tissue from fetuses in whom CHB led to death, and by determination of the levels of erythropoietin in the cord blood of anti-SSA/Ro–exposed CHB-affected and unaffected neonates. Fibroblasts isolated from human fetal hearts and lungs (a nonsusceptible organ) were cultured under conditions of hypoxia and environmental modulation of cAMP, and evaluated for expression of messenger RNA (mRNA) and protein related to homeostasis of extracellular fibrous matrix.
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To date, a reasonable explanation for the rarity of CHB remains a challenging research issue. While substantial evidence exists that anti-SSA/Ro antibodies are a necessary factor, the discordance of disease in identical twins is an observation difficult to reconcile. Given the consideration that a stressful in utero environment might differentially affect one fetus versus another, hypoxia was addressed as an accelerating factor that might tip the balance from vulnerability to full expression of disease.
Based on the intriguing observations of rapid scarring of the AV node, the concomitant or even isolated findings of endocardial fibroelastosis, and the susceptibility of the heart to CHB compared with other organs, we selected fetal cardiac and lung fibroblasts as the focus of this study. The results presented herein support hypoxia as an environmental stress that potentially affects the distal component of the pathologic cascade. In keeping with our own hypothesis previously reported (23), and with genetic studies on the increased frequency of a profibrosing TGFβ polymorphism in CHB-affected fetuses (24, 25), it is envisioned that hypoxia may amplify the injurious effects of scarring cytokines, such as TGFβ, that are secreted by infiltrating macrophages during the clearance of opsonized anti-SSA/Ro–bound cardiocytes.
It is particularly challenging to provide proof of concept with regard to the hypoxia hypothesis, because in only 20% of fetuses CHB is fatal, and in these cases, death is often weeks after the detection of bradycardia, making it difficult to identify relevant pathologic footprints. The availability of an electively terminated fetal heart for immunohistologic evaluation within days of the echocardiographic diagnosis of CHB (20 weeks) and another fetus dying close to the time of diagnosis (22 weeks) provided us with a rare opportunity to assess very proximate biologic events. Although it is fully acknowledged that a decrease in cardiac output itself could be the cause of hypoxia, in the electively terminated fetus studied herein there were no signs of low cardiac output due to myocardial dysfunction at the detection of CHB.
The accumulation of HIF-1α, a well-characterized transcription factor complex that regulates hypoxia-driven gene expression (26, 27), may represent a causative factor. In addition, expression of mTOR was recently advanced as a mediator of fibrosis in scleroderma (17). Notably, the HIF-1α and mTOR were localized to regions in which we have previously observed TGFβ staining and nuclear translocation of SMAD2 (3). Expression of HIF-1α in the second CHB case is more difficult to interpret, since this fetus had a severe cardiomyopathy that resulted in death within 2 weeks of the diagnosis of CHB; therefore, attribution of hypoxia to the initial injury is more ambiguous. HIF-1α was not detected in the electively terminated normal heart. Testing the amplifying effect of hypoxia may be of interest in an animal model, since attempts to generate anti-SSA/Ro–mediated heart block in pups has yielded observations of predominantly first-degree block (28) and a low penetrance of third-degree block (28–30).
A hypoxic stimulus results in an increase in erythropoietin levels, to enhance red cell oxygen-carrying capacity. Cord levels of this glycoprotein, which does not cross the placenta, are an indicator of tissue oxygenation and reflect production solely by the fetus. In contrast to umbilical arterial pH changes, which reflect acute hypoxia, elevations of erythropoietin are considered to indicate prolonged fetal hypoxia. Relevant to the timing of CHB, increased fetal production of erythropoietin has been reported to occur from 20 weeks' gestation (31). Thus, elevated cord blood levels could represent ongoing hypoxic conditions that were initiated months earlier and have been sustained.
In consideration of erythropoietin as a biomarker of hypoxia, it is notable that in several studies, a negative association with birth weight has been observed (32). Pertinent to the consideration of hypoxia as an amplifying factor in CHB (as well as the discordance of disease in twins exposed to identical maternal antibodies) is the report that in 5 sets of twins (not related to neonatal lupus) in whom birth weights differed by more than 25%, the mean erythropoietin concentration was significantly higher in the smaller twin than in the larger twin (32). Arguably, in the situation of CHB, it is equally plausible that hypoxia was not causal but, rather, a consequence of the disease once established. However, based on the data presented herein, CHB per se does not invariably increase the cord blood level of erythropoietin. This makes sense, since hypoxia is hypothesized to be an amplifying, but not requisite, factor in the pathogenesis of CHB. Given a sufficiently vigorous inflammatory component, scarring would be predicted to proceed in normoxic conditions. In the absence of fetal sampling during gestation (impractical for research purposes), the finding of elevated cord blood levels of erythropoietin conceptually supports the in vitro findings presented herein, but should be interpreted with consideration of potential confounders.
Adrenomedullin, a 52–amino-acid vasodilator protein that up-regulates cAMP in target cells including fibroblasts (21, 33), was identified via the array approach. Adrenomedullin has previously been reported to be synthesized by endothelial cells, healthy renal cells, and renal carcinoma cells exposed to hypoxia (34–36). The increased transcription of adrenomedullin following exposure to hypoxia provided the link to cAMP, but generated a potential paradox in that the net effect of hypoxia was to augment fibrosis. Nguyen and Claycomb demonstrated that in adult rat ventricular cardiac myocytes, hypoxia induced transcription of the adrenomedullin genes by HIF-1α (37). Findings suggestive of the protective effects of endogenous adrenomedullin on cardiac fibrosis were obtained in mice heterozygous for an adrenomedullin-null mutation, since left ventricular wall thickness and perivascular fibrosis following aortic constriction were worse in these mice compared with wild-type mice (38). One plausible explanation for the results reported herein is that in the human fetal cardiac fibroblast, adrenomedullin is expressed in an attempt to protect against fibrosis, but is ineffective in repletion of sufficient cAMP.
With regard to the effects of both hypoxia and TGFβ, changes in cAMP levels appear to be highly influential in the regulation of cardiac fibroblast transdifferentiation. Transformation of cardiac fibroblasts to myofibroblasts, characterized by expression of SMA and the production of extracellular matrix components, is a pivotal event in the remodeling of connective tissue. Exposure of human fetal cardiac fibroblasts to hypoxia resulted in the expression of a scarring phenotype, equivalent to the effect observed after coincubation with TGFβ. Raising the level of cAMP inhibited this effect. In support of these findings, Swaney et al recently reported that increased cAMP levels modulate the transformation of adult rat cardiac fibroblasts to myofibroblasts; specifically, forskolin inhibited TGFβ-induced SMA expression and collagen synthesis (21).
One theory for the resistance to a scarring phenotype that has been observed in the pulmonary fibroblast is that this organ is in a state of chronic hypoxia, given that blood flow to the lungs is not important until birth. These findings suggest that pleiotropic responses are cell-type–specific, as has been previously reported (39). Perhaps differential susceptibility to changes in cAMP accounts, in part, for the predominance of the heart as the target organ injured by maternal anti-SSA/Ro antibodies. Thus, in addition to an inflammatory injury, the inherent inability of cardiac tissue to maintain oxygen homeostasis when challenged may tip the balance toward scar formation.
It is readily acknowledged that translation of our in vitro findings to human pregnancy and CHB is limited by the available data. However, hypoxia does occur during pregnancy and can originate either in the mother or at the level of the placenta, since oxygen exchange between the mother and fetus occurs via the placenta. Although the generally accepted causes of maternal hypoxia, such as hypertension, diabetes, infection, anemia, and smoking, have not been systematically evaluated during pregnancies in which the anti-SSA/Ro–positive mother has a fetus with a diagnosis of CHB, changes in oxygen during the period of vulnerability (at 16–24 weeks' gestation) appear to be plausible.
Our data thus support the novel hypothesis that hypoxia is a potential environmental stress factor that is capable of amplifying the myofibroblast transdifferentiation of a fetal cardiac fibroblast that has already been subjected to an inflammatory injury initiated by circulating maternal anti-SSA/Ro antibodies. In the process of normal repair, the fibroblast is expected to mediate a role involving matrix synthesis that is associated with reversible wounding without scar formation. In this scenario, hypoxia causes an increase in HIF-1α, which induces the expression of SMA, but at the same time increases cAMP. Thus, hypoxia under physiologic circumstances promotes a matrix-promoting process and a molecule that restrains this process. In a pathologic scenario, the environment (e.g., hypoxia) promotes exuberant matrix synthesis that is irreversible (a fibrotic process). This may occur in the CHB-affected fibroblast, which is primed by antecedent inflammatory events and already expressing HIF-1α, as evidenced by histologic findings. Increasing focus on the causes and prevention of abnormal oxygen homeostasis may provide a new direction for research on the pathogenesis of CHB.