Role of hypoxia and cAMP in the transdifferentiation of human fetal cardiac fibroblasts: implications for progression to scarring in autoimmune-associated congenital heart block.
OBJECTIVE
Identification of isolated congenital heart block (CHB) predicts, with near certainty, the presence of maternal anti-SSA/Ro antibodies; however, the 2% incidence of CHB in first offspring of anti-SSA/Ro+ mothers, 20% recurrence in subsequent pregnancies, and discordance in identical twins suggest that an environmental factor amplifies the effect of the antibody. Accordingly, this study was carried out to explore the hypothesis that hypoxia potentiates a profibrosing phenotype of the fetal cardiac fibroblast.


METHODS
Evidence of an effect of hypoxia was sought by immunohistologic evaluation of CHB-affected fetal heart tissue and by determination of erythropoietin levels in cord blood. The in vitro effect of hypoxia on gene expression and phenotype in fibroblasts derived from fetal hearts and lungs was investigated by Affymetrix arrays, quantitative polymerase chain reaction (PCR), immunofluorescence, and immunoblotting.


RESULTS
In vivo hypoxic exposure was supported by the prominent intracellular fibroblast expression of hypoxia-inducible factor 1alpha in conduction tissue from 2 fetuses in whom CHB led to death. The possibility that hypoxia was sustained was suggested by significantly elevated erythropoietin levels in cord blood from CHB-affected, as compared with unaffected, anti-SSA/Ro-exposed neonates. In vitro exposure of cardiac fibroblasts to hypoxia resulted in transdifferentiation to myofibroblasts (a scarring phenotype), as demonstrated on immunoblots and immunofluorescence by increased expression of smooth muscle actin (SMA), an effect not seen in lung fibroblasts. Hypoxia-exposed cardiac fibroblasts expressed adrenomedullin at 4-fold increased levels, as determined by Affymetrix array, quantitative PCR, and immunofluorescence, thus focusing attention on cAMP as a modulator of fibrosis. MDL12,330A, an adenylate cyclase inhibitor that lowers the levels of cAMP, increased expression of fibrosis-related proteins (mammalian target of rapamycin, SMA, plasminogen activator inhibitor type 1, and type I collagen), while the cAMP activator forskolin attenuated transforming growth factor beta-elicited fibrosing end points in the cardiac fibroblasts.


CONCLUSIONS
These findings provide evidence that hypoxia may amplify the injurious effects of anti-SSA/Ro antibodies. Modulation of cAMP may be a key component in the scarring phenotype. Further assessment of the susceptibility of cardiac fibroblasts to cAMP modulation offers a new research direction in CHB.
Clancy
Robert M
RM
Department of Medicine, Division of Rheumatology, New York University School of Medicine, 560 First Avenue, New York, NY 10016, USA. bobdclancy@aol.com
Zheng
Ping
P
O'Mahony
Marguerita
M
Izmirly
Peter
P
Zavadil
Jiri
J
Gardner
Lawrence
L
Buyon
Jill P
JP
D000199:Actins / Q000378:metabolism; D053607:Adrenomedullin / Q000378:metabolism; D000974:Antibodies, Antinuclear / Q000378:metabolism; D015551:Autoimmunity / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology*; D054338:Cell Transdifferentiation / Q000502:physiology*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D018450:Disease Progression; D004921:Erythropoietin / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology*; D005355:Fibrosis / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D006321:Heart / Q000196:embryology*; D006327:Heart Block / Q000235:genetics* / Q000276:immunology* / Q000473:pathology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D008168:Lung / Q000196:embryology / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D000199:Actins; D000974:Antibodies, Antinuclear; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; C035356:SS-A antibodies; D016212:Transforming Growth Factor beta; D004921:Erythropoietin; D053607:Adrenomedullin; D000242:Cyclic AMP