Background/Purpose: Maternal autoantibodies (Ab) reactive with the Ro/La ribonucleoprotein complex are associated with the development of cardiac injury in a fetus passively exposed to these Ab. This study evaluated the irreversible scarring phenotype characteristic of heart block involving the mitogen activated protein kinase (MAPK) pathway and its regulation by cAMP.

Methods: The aorta from a healthy 2^nd trimester fetal heart was cannulated using a Langendorff preparation with the addition of proteolytic enzymes to yield a single cell suspension of primary human fetal cardiac fibroblasts. Cultured cells were treated with secreted products generated from activated macrophages with or without BIX 02189 (10uM, a specific MEK5/ERK5 inhibitor) and forskolin (10uM to raise cAMP). After RNA isolation and cDNA library preparation, RNA-Seq, transcriptome analysis (Data as log2 transcripts per million) and qPCR were performed.

Results: Incubation of fibroblasts with supernatants from macrophages transfected with hY3 (ssRNA associated with Ro60), shown to induce a pro-fibrotic phenotype, resulted in the increased expression of 3836 genes. Based on DAVID functional annotation, the top clusters represented were Actin Binding, Cytoskeletal Protein Binding, Cell Adhesion, Signal Peptide, and Contractile Fiber, all processes considered typical of the myofibroblast phenotype. In addition, RAPGEF3, an endogenous ERK5 inhibitor, and Adrenomedullin, which increases cAMP, were downregulated while PDE4D, an inhibitor of cAMP generation, was upregulated (Table 1). These data are consistent with previous literature supporting the association of lowered intracellular cAMP and upregulation of pro-fibrotic genes. Given that cAMP attenuates the activity of ERK5, BIX 02189 was used to evaluate the transcriptome. Of the 3836 genes upregulated by hY3 macrophage supernatants, 617 were reversed by the subsequent addition of BIX. Among the upregulated genes were pro-fibrosing genes such as EDN1 and TGFβ2 and among those downregulated were genes that resist fibrosis including CLU, RAPGEF3, and ADM. The latter two are associated with a cAMP dependent inhibition of ERK5 and increased cAMP, respectively. The pro-fibrosing EDN1 result was confirmed by qPCR and as expected was attenuated by forskolin.

Conclusion: These data support that the link of anti-Ro immune complex activated macrophages and the pathogenic fibroblast phenotype may relate to a decrease in cAMP levels. These results highlight potential novel targets for therapy and solidify the role of ERK5 in the transdifferentiation of fetal fibroblasts in the context of congenital heart block.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cyclic-amp-erk5-and-transdifferentiation-of-cardiac-fibroblasts-in-the-pathogenesis-of-autoimmune-congenital-heart-block/