Epicardial differentiation drives fibro-fatty remodeling in arrhythmogenic cardiomyopathy
Kohela, Arwa; van Kampen, Sebastiaan J; Moens, Tara; Wehrens, Martijn; Molenaar, Bas; Boogerd, Cornelis J; Monshouwer-Kloots, Jantine; Perini, Ilaria; Goumans, Marie José; Smits, Anke M; van Tintelen, J Peter; van Rooij, Eva
(2021) Science translational medicine, volume 13, issue 612, pp. 1 - 15
(Article)
Abstract
Arrhythmogenic cardiomyopathy (ACM) is an inherited disorder often caused by pathogenic variants in desmosomal genes and characterized by progressive fibrotic and fat tissue accumulation in the heart. The cellular origin and responsible molecular mechanisms of fibro-fatty deposits have been a matter of debate, due to limitations in animal models recapitulating
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this phenotype. Here, we used human-induced pluripotent stem cell (hiPSC)–derived cardiac cultures, single-cell RNA sequencing (scRNA-seq), and explanted human ACM hearts to study the epicardial contribution to fibro-fatty remodeling in ACM. hiPSC-epicardial cells generated from patients with ACM showed spontaneous fibro-fatty cellular differentiation that was absent in isogenic controls. This was further corroborated upon siRNA-mediated targeting of desmosomal genes in hiPSC-epicardial cells generated from healthy donors. scRNA-seq analysis identified the transcription factor TFAP2A (activating enhancer-binding protein 2 alpha) as a key trigger promoting this process. Gain- and loss-of-function studies on hiPSC-epicardial cells and primary adult epicardial-derived cells demonstrated that TFAP2A mediated epicardial differentiation through enhancing epithelial-to-mesenchymal transition (EMT). Furthermore, examination of explanted hearts from patients with ACM revealed epicardial activation and expression of TFAP2A in the subepicardial mesenchyme. These data suggest that TFAP2A-mediated epicardial EMT underlies fibro-fatty remodeling in ACM, a process amenable to therapeutic intervention.
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Keywords: General Medicine, Journal Article
ISSN: 1946-6234
Publisher: American Association for the Advancement of Science
Note: Funding Information: Human PKP2 c.2013delC, PKP2 c.1849C>T, and control hiPSC lines were provided by H.-S. V. Chen at University of California San Diego (29), J. Wu at Stanford Cardiovascular Institute (supported by National Institutes of Health R24 HL117756), and M. Bellin and C. Freund at Leiden University Medical Center (41), respectively. hiPSCs were maintained in Essential 8 Medium (Thermo Fisher Scientific, A1517001) on Geltrex (Thermo Fisher Scientific, A1413302)–coated plates (42). The hiPSC-epicardial cell differentiation protocol was adapted and optimized from Bao et al. (16). Briefly, hiPSCs were cultured until confluent in Essential 8 Medium on Geltrex-coated flasks. On day 0, medium was replaced with differentiation medium [RPMI medium (Thermo Fisher Scientific, 72400021) containing l-ascorbic acid (0.2 mg/ml; Sigma-Aldrich, A8960)] and supplied with 4 M CHIR99021 (Sigma-Aldrich, SML1046) to promote mesoderm differentiation. On day 2, medium was replaced with differentiation medium containing 5 M IWP2 (Inhibitor of Wnt Production-2) (Millipore, 681671) to direct cardiac progenitor cell differentiation, which was then changed to differentiation medium alone on day 4. Cardiac progenitor cells were generated on day 6 at which point cells were dissociated using StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific, A1110501) containing 2.5% trypsin (Thermo Fisher Scientific, 15090046) and subsequently cultured in LaSR basal medium [Advanced Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Thermo Fisher Scientific, 12634010) containing l-ascorbic acid (0.1 mg/ml; Sigma-Aldrich, A8960)]. On day 7, medium was replaced with LaSR basal medium containing 4 M CHIR99021 to promote epicardial cell fate. Nonaddition of CHIR99021 at day 7 promoted a cardiomyocyte cell lineage. On day 9, medium was changed to LaSR basal medium. From day 12 onward, cells were maintained in LaSR basal medium containing TGF inhibitor (Stemgent, 04-0014) to prevent spontaneous EMT and split whenever confluent. Thiazovivin (2 M; Millipore, 420220) was added at every cell split. The hiPSC-cardiomyocyte differentiation protocol was adapted from Burridge et al. (42). Briefly, cells were treated similarly to hiPSC-epicardial cells up to day 4. On day 6, cells were refreshed with differentiation medium for 2 days. On day 8 and every 2 to 3 days thereafter, medium was changed to cardio culture medium [RPMI medium (Thermo Fisher Scientific, 72400021) containing B-27 supplement (Thermo Fisher Scientific, 17504001)]. Around day 14, cells were replated at a lower density in cardio culture containing 2 M thiazovivin and subsequently cultured in cardio selection medium [RPMI no glucose (Biological Industries 01-101-1A), 4 mM lactate-Hepes, recombinant human albumin (0.5 mg/ml), and l-ascorbic acid (0.2 mg/ml)] for 4 days. Cells were subsequently maintained in cardio culture medium. Funding Information: We thank H. V. Chen, J. C. Wu, M. Bellin, and C. Freund for providing the hiPSC lines and Y. Post for technical support. This work was supported by the Dutch CardioVascular Alliance (DCVA), an initiative with support of the Dutch Heart Foundation (DCVA2017-18 ARENA-PRIME to E.v.R. and 2015-30 eDETECT and 2018-30 PREDICT2 to J.P.v.T.). C.J.B. received funding from the European Union?s Horizon 2020 research and innovation program under the Marie Sk?odowska-Curie grant agreement no. 751988. Publisher Copyright: Copyright © 2021 The Authors, some rights reserved.
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