The identification of congenital heart block (CHB) in a fetus, particularly in the late second trimester and in the absence of structural abnormalities, predicts with at least 85% certainty that the mother will have autoantibodies to components of the intracellular SSA/Ro–SSB/La system (1). With increasing awareness of this association, it has become evident that the spectrum of cardiac injury not only includes first- to third-degree block, but can also extend to the myocardium and endocardium (2). The pathologic cascade to irreversible fibrosis, characteristic of autoimmune-associated CHB, has been difficult to define at the molecular level. The challenge rests on integrating the initial antibody insult with the final cardiac injury and reconciling the fact that most infants born to mothers with the candidate antibodies do not have clinically detectable atrioventricular (AV) block (3). The pathway to scarring may be variable: kept totally in check in most fetuses (normal sinus rhythm), remaining subclinical in others (first-degree block), and becoming fully executed in a very few (advanced block). Moreover, CHB is an injury unique to some phase(s) of development, since it has never been reported in the maternal heart despite the presence of identical antibodies in the maternal circulation.
In vitro studies employing cardiac myocytes and fibroblasts separately isolated and cultured from human fetal hearts provide evidence for a pathologic link between antibodies and injury (4). After induction of apoptosis, SSA/Ro and SSB/La translocate from their normal intracellular residence to the surface of apoptotic blebs, where they can be bound by their cognate antibodies and become opsonized (5, 6). Cocultured macrophages have been shown to phagocytose opsonized apoptotic cardiocytes and to secrete inflammatory cytokines such as tumor necrosis factor α (TNFα) (6). Other investigators have also demonstrated that phagocytosis of opsonized apoptotic cells is proinflammatory (7, 8), as exemplified by the observation that ingestion of apoptotic cells bound by anticardiolipin antibodies results in the release of TNFα from cocultured macrophages (8). Human fetal cardiac fibroblasts exposed to supernatants obtained from macrophages incubated with opsonized apoptotic cardiocytes markedly increased expression of the transdifferentiation myofibroblast marker smooth muscle actin (SMA), associated with scarring, an effect blocked by neutralizing anti–transforming growth factor β (anti-TGFβ) antibodies (4).
Validation of the in vitro model described above has been limited by the availability of histologic specimens from hearts of fetuses dying of CHB, since the mortality rate is relatively low (2) and death most often occurs weeks to months after initial detection of bradyarrhythmia. However, immunohistologic study of the heart of a full-term male infant first diagnosed as having AV block at age 19 weeks supported the notion of macrophage crosstalk, despite a 5-month interval from detection to death (4). Ventricular tissue revealed microcalcification in which a predominant SMA-positive infiltrate was seen. Macrophages were also observed in areas of scar tissue. Notably, the fibrosis was not bland, but involved an infiltrate of activated myofibroblasts months after the initial insult. To confirm and extend these findings, this and 3 other hearts, encompassing a spectrum of disease severity and timing of death relative to clinical detection, were extensively examined. The present study represents a first-time evaluation of each component of the proposed pathologic cascade to scarring. Accordingly, the focus was on the extent of apoptosis, IgG deposition, macrophage accumulation, and fibroblast phenotype.
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- PATIENTS AND METHODS
Defining the pathologic scenario that eventually results in fibrotic replacement of the AV node and, in some cases, more extensive damage to the myocardium and endocardium, has relied on in vitro studies. Histologic evaluation has been limited not only by the rarity of the disease, but also by the fact that death, if it occurs at all, most often happens weeks to months after bradycardia is first identified.
The present study is the first to evaluate the extent of apoptosis and its relationship to immunoglobulin deposition, cellular infiltration, and fibroblast transdifferentiation in cardiac tissue from fetuses dying at various time points after the clinical diagnosis of heart block. The availability of two fetal hearts almost immediately after the diagnosis of a conduction defect facilitated a window of opportunity to capture IgG deposition in close proximity to apoptotic cells and macrophages. The accumulation of these inflammatory cells and the transdifferentiation of fibroblasts into myofibroblasts (the cellular engines driving calcification and fibrosis) were prominent features in the fetuses that died earliest after diagnosis. That fibrosis was already present by the time heart block was documented strongly suggests that the cascade leading to scarring proceeds rapidly and that reversing it will be difficult. The study of cardiac tissue from a fetus dying months after the initial diagnosis of bradycardia provides insights into the sustained nature of the disease process. Persistence of the myofibroblasts may contribute to the postnatal progression of disease in the cases of incomplete blocks at birth (11), as well as to the development of endocardial fibroelastosis (12) and life-threatening cardiomyopathies (13).
The exaggerated apoptosis observed in the hearts from fetuses with CHB/myocarditis is consistent with the notion that this specialized form of cell death plays a key role in the initiation of an inflammatory response. The highest levels of apoptosis were observed in septal regions containing conduction tissue. One plausible explanation for this increased apoptosis is derived from in vitro studies demonstrating that macrophages secrete cytokines such as TNF and TGF following phagocytosis of opsonized cardiocytes (4, 6). Recent studies have demonstrated that TNF and TGF contribute to cell-cycle regulation of cells derived from mesenchymal lineage, indicating that these cytokines may promote apoptosis of human fetal cardiocytes in CHB (14, 15). These observations support the notion that one candidate fetal factor conferring increased susceptibility to permanent cardiac injury might relate to exaggerated apoptosis, perhaps via increased secretion of TNF. This is supported by preliminary data demonstrating an increased frequency of the high-secreting −308A allele (TNF2) of TNF in children with neonatal lupus and their healthy siblings compared with that in population controls (16).
The finding of IgG in close proximity to the apoptotic cells extends previous research demonstrating that transplacentally trafficked anti-SSB/La antibodies bind apoptotic cells in the murine fetal heart (17). However, in the present study, it is fully acknowledged that the IgG specificity is unknown and awaits identification by antiidiotypes, an approach which must take into account the possibility of private idiotypes. Although apoptosis had not been previously examined, earlier studies of fetuses dying of CHB and hydrops (at 29 and 30 weeks of gestation) showed IgG deposition in several areas of the heart including the conduction system (18, 19). The investigators reported that, in some areas, “IgG appeared to outline cells” (19). In a study of endocardial fibroelastosis, Nield et al (12) demonstrated IgG deposition in the RV, LV, and AV nodal region in several cases of CHB. However, in a fetus with endocardial fibroelastosis without CHB whose mother was anti-SSB/La positive, apoptosis was not identified (20). Horsfall et al (21) demonstrated IgG binding to the surface of myocardial fibers in a fatal case of CHB and further identified the target antigen as SSB/La using a maternal anti-SSB/La idiotype.
The notion of an inflammatory component in the cascade to cardiac fibrosis was supported by the demonstration of macrophages and multinucleated giant cells, and this finding extends the previous report of a mononuclear cell infiltration in the myocardium of a fetus dying in utero at 18 weeks of gestation (22), as well as the previously reported demonstration of patchy lymphoid aggregates throughout the myocardium of an infant delivered at 30 weeks and dying in the immediate postnatal period (19). Macrophages potentially contribute to several aspects of the pathologic process mediated by maternal autoantibodies. Although the pathways of clearance and cytokine secretion may vary, macrophages phagocytose both nonopsonized and opsonized apoptotic cells, the former via phosphatidylserine receptors, which is generally considered a noninflammatory process (23). In CHB, macrophages may provide a critical link between inflammation and ultimate scarring by secretion of APs resulting in increased calcification (24). In fact, macrophages could be seen contained in areas of calcification, particularly in the early cases. However, in the full-term neonate who died at birth, macrophages were less abundant and not associated with calcified areas, suggesting a diminished role in inflammation as the pathologic cascade progresses.
Abnormal stimulation of the resident fibroblasts by macrophages probably constitutes a means by which fibrotic sequelae are further amplified. Indeed, the histologic sections demonstrated the novel finding of macrophage clusters in close proximity to myofibroblasts in scar tissue near the AV groove as well as the thickened fibrous subendocardium. The functional implication of this cellular colocalization is suggested by in vitro studies in which cultured human fetal cardiac fibroblasts, exposed to supernatants obtained from macrophages incubated with opsonized apoptotic cardiocytes, had markedly increased expression of the myofibroblast marker SMA (scarring phenotype) (4). The addition of neutralizing anti-TGF antibodies to the “opsonized” supernatant blocked expression of SMA, supporting a potential role of TGF in the final pathologic cascade to scarring. Of relevance, preliminary genotyping data suggest that children with CHB have a higher frequency of the fibrosis-promoting polymorphism at codon 10 of TGF than do their unaffected siblings (16).
The unambiguous demonstration of macrophages and myofibroblasts in all the affected cases substantiates the pathologic crosstalk described in reports of our earlier in vitro studies (4). The fibrosis observed was not bland, but involved infiltration of activated myofibroblasts as long as 5 months after the initial insult. The dedifferentiation of fibroblasts to myofibroblasts is associated with scar formation. Fibrosis is due to a persistent myofibroblast, a phenotype associated with “wounding.” While it is often assumed that fibrosis is simply the end result of an inflammatory insult, a recent report of Lyme carditis with second-degree heart block (25) prompts a reappraisal of the elements of tissue injury, response, and ultimate repair or scar in the human heart. Right ventricular biopsy revealed mononuclear cells around the myocardial microvasculature and within the endocardium. Despite prolonged inflammation (heart block present for 8 weeks), the cascade to fibrosis was not irrevocably programmed, since sinus rhythm was restored following antibiotic therapy. This absence of permanent injury stands in strong contrast to the rapid progression to scarring seen in autoantibody-associated CHB. The expression of specific combinations of cytokines may ultimately provide the explanation.
In summary, immunohistologic findings in available cardiac sections from several cases of CHB/myocarditis with varying degrees of pathology parallel the results obtained exploiting in vitro coculturing systems. Physiologic apoptosis may initiate an inflammatory process via antibody binding and ingestion by macrophages, which not only fuels continued apoptosis but also contributes to the transdifferentiation of cardiac fibroblasts to a scarring phenotype. Persistence of this phenotype even after birth may be related to the progression of block seen in some infants postpartum. The heart block of neonatal lupus is not only progressive (second to third degree) but also characteristically irreversible, despite brief exposure to autoantibodies and limited period of inflammation. Moreover, fibrosis of the AV node contradicts the paradigm that fetal wounds heal without scarring (26). Disruption of healing may involve the continued presence of myofibroblasts, a consequence of protracted stimulation from the macrophages. Irreversible fibrotic replacement of normal tissue may be unique to heart block acquired in utero following autoantibody-initiated inflammation. Other inflammatory stimuli, as in Lyme disease, induce transient block (25), which is evidence against the assumption that fibroblast transdifferentiation is merely a common final pathway of inflammation.