Role of hypoxia and cAMP in the transdifferentiation of human fetal cardiac fibroblasts: Implications for progression to scarring in autoimmune‐associated congenital heart block

Abstract

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 1␣ 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 CHBaffected, as compared with unaffected, anti-SSA/Roexposed 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 ␤-elicited fibrosing end points in the cardiac fibroblasts.Conclusion. 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.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

Hypoxic Stress Induces Transient Receptor Potential Melastatin 2 (TRPM2) Channel Expression in Adult Rat Cardiac Fibroblasts

“…Hypoxia is known to induce differentiation from fibroblasts to myofibroblasts (6). However, mRNA levels of myofibroblast markers, such as TGF-β1, p21, and α-SMA, were not increased in our hypoxia-exposed cells.…”

“…Therefore, we next examined the mRNA expression levels of TRPM2 channels and KCa3.1 in cardiac fibroblasts exposed to hypoxia and normoxia. In addition, we also examined the expression levels of marker mRNAs for myofibroblasts, for example, transforming growth factor (TGF)-β1 (24), cyclin-dependent kinase inhibitor 1 (p21), and α-smooth muscle actin (α-SMA) (25), because hypoxia is known to induce differentiation from fibroblasts to myofibroblasts (6). As illustrated in Fig.…”

“…Cardiac fibroblasts play an important role in this process. For example, hypoxia triggers the differentiation of fibroblasts to myofibroblasts (6), leading to their matrix metalloprotease (MMP)-dependent invasion (7) with collagen synthesis (8) and resulting cardiac remodeling. Thus, hypoxia is a pathophysiological stimulus for cardiac fibroblasts.…”

The protective effect of ursodeoxycholic acid in an in vitro model of the human fetal heart occurs via targeting cardiac fibroblasts

“…It can therefore be speculated that the relative state of chronic hypoxia in which the human heart develops is necessary for the normal development of the cardiovascular system (Webster & Abela, 2007). Clancy et al (2007) showed that hypoxia is thought to contribute to differentiation of human fetal cardiac fibroblasts into myofibroblasts in vitro (Clancy et al, 2007). It has also been postulated that fetal death in ICP may be mediated by bile acid induced vasoconstriction of placental vessels leading to acute hypoxia in these fetuses (Mays, 2010;Sepúlveda et al, 1991).…”

“…The effect of hypoxia on fibroblasts is attenuated by UDCA A fetus develops in a state of partial hypoxia during pregnancy (Clancy et al, 2007;Patterson and Zhang, 2010;Webster and Abela, 2007). Therefore, further investigation into the effect of hypoxia on neonatal rat and human fetal fibroblasts was carried out.…”