Ping Yuan1,2, Sirisha M Cheedipudi1, Leila Rouhi1, Siyang Fan1, Lukas Simon3, Zhongming Zhao3, Kui Hong2, Priyatansh Gurha1, Ali J Marian1. 1. Center for Cardiovascular Genetics, Institute of Molecular Medicine and Department of Medicine (P.Y., S.M.C., L.R., S.F., P.G., A.J.M.). 2. Department of Cardiovascular Medicine, Second Affiliated Hospital of Nanchang University, China (P.Y., K.H.). 3. Center for Precision Health, School of Biomedical Informatics and School of Public Health, University of Texas Health Science Center at Houston (L.S., Z.Z.).
Abstract
BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) manifests with sudden death, arrhythmias, heart failure, apoptosis, and myocardial fibro-adipogenesis. The phenotype typically starts at the epicardium and advances transmurally. Mutations in genes encoding desmosome proteins, including DSP (desmoplakin), are major causes of ACM. METHODS: To delineate contributions of the epicardium to the pathogenesis of ACM, the Dsp allele was conditionally deleted in the epicardial cells in mice upon expression of tamoxifen-inducible Cre from the Wt1 locus. Wild type (WT) and Wt1-CreERT2:DspW/F were crossed to Rosa26mT/mG (R26mT/mG) dual reporter mice to tag the epicardial-derived cells with the EGFP (enhanced green fluorescent protein) reporter protein. Tagged epicardial-derived cells from adult Wt1-CreERT2:R26mT/mG and Wt1-CreERT2: R26mT/mG:DspW/F mouse hearts were isolated by fluorescence-activated cell staining and sequenced by single-cell RNA sequencing. RESULTS: WT1 (Wilms tumor 1) expression was progressively restricted postnatally and was exclusive to the epicardium by postnatal day 21. Expression of Dsp was reduced in the epicardial cells but not in cardiac myocytes in the Wt1-CreERT2:DspW/F mice. The Wt1-CreERT2:DspW/F mice exhibited premature death, cardiac dysfunction, arrhythmias, myocardial fibro-adipogenesis, and apoptosis. Single-cell RNA sequencing of ≈18 000 EGFP-tagged epicardial-derived cells identified genotype-independent clusters of endothelial cells, fibroblasts, epithelial cells, and a very small cluster of cardiac myocytes, which were confirmed on coimmunofluorescence staining of the myocardial sections. Differentially expressed genes between the paired clusters in the 2 genotypes predicted activation of the inflammatory and mitotic pathways-including the TGFβ1 (transforming growth factor β1) and fibroblast growth factors-in the epicardial-derived fibroblast and epithelial clusters, but predicted their suppression in the endothelial cell cluster. The findings were corroborated by analysis of gene expression in the pooled RNA-sequencing data, which identified predominant dysregulation of genes involved in epithelial-mesenchymal transition, and dysregulation of 146 genes encoding the secreted proteins (secretome), including genes in the TGFβ1 pathway. Activation of the TGFβ1 and its colocalization with fibrosis in the Wt1-CreERT2:R26mT/mG:DspW/F mouse heart was validated by complementary methods. CONCLUSIONS: Epicardial-derived cardiac fibroblasts and epithelial cells express paracrine factors, including TGFβ1 and fibroblast growth factors, which mediate epithelial-mesenchymal transition, and contribute to the pathogenesis of myocardial fibrosis, apoptosis, arrhythmias, and cardiac dysfunction in a mouse model of ACM. The findings uncover contributions of the epicardial-derived cells to the pathogenesis of ACM.
BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) manifests with sudden death, arrhythmias, heart failure, apoptosis, and myocardial fibro-adipogenesis. The phenotype typically starts at the epicardium and advances transmurally. Mutations in genes encoding desmosome proteins, including DSP (desmoplakin), are major causes of ACM. METHODS: To delineate contributions of the epicardium to the pathogenesis of ACM, the Dsp allele was conditionally deleted in the epicardial cells in mice upon expression of tamoxifen-inducible Cre from the Wt1 locus. Wild type (WT) and Wt1-CreERT2:DspW/F were crossed to Rosa26mT/mG (R26mT/mG) dual reporter mice to tag the epicardial-derived cells with the EGFP (enhanced green fluorescent protein) reporter protein. Tagged epicardial-derived cells from adult Wt1-CreERT2:R26mT/mG and Wt1-CreERT2: R26mT/mG:DspW/F mouse hearts were isolated by fluorescence-activated cell staining and sequenced by single-cell RNA sequencing. RESULTS: WT1 (Wilms tumor 1) expression was progressively restricted postnatally and was exclusive to the epicardium by postnatal day 21. Expression of Dsp was reduced in the epicardial cells but not in cardiac myocytes in the Wt1-CreERT2:DspW/F mice. The Wt1-CreERT2:DspW/F mice exhibited premature death, cardiac dysfunction, arrhythmias, myocardial fibro-adipogenesis, and apoptosis. Single-cell RNA sequencing of ≈18 000 EGFP-tagged epicardial-derived cells identified genotype-independent clusters of endothelial cells, fibroblasts, epithelial cells, and a very small cluster of cardiac myocytes, which were confirmed on coimmunofluorescence staining of the myocardial sections. Differentially expressed genes between the paired clusters in the 2 genotypes predicted activation of the inflammatory and mitotic pathways-including the TGFβ1 (transforming growth factor β1) and fibroblast growth factors-in the epicardial-derived fibroblast and epithelial clusters, but predicted their suppression in the endothelial cell cluster. The findings were corroborated by analysis of gene expression in the pooled RNA-sequencing data, which identified predominant dysregulation of genes involved in epithelial-mesenchymal transition, and dysregulation of 146 genes encoding the secreted proteins (secretome), including genes in the TGFβ1 pathway. Activation of the TGFβ1 and its colocalization with fibrosis in the Wt1-CreERT2:R26mT/mG:DspW/F mouse heart was validated by complementary methods. CONCLUSIONS: Epicardial-derived cardiac fibroblasts and epithelial cells express paracrine factors, including TGFβ1 and fibroblast growth factors, which mediate epithelial-mesenchymal transition, and contribute to the pathogenesis of myocardial fibrosis, apoptosis, arrhythmias, and cardiac dysfunction in a mouse model of ACM. The findings uncover contributions of the epicardial-derived cells to the pathogenesis of ACM.
Authors: Estelle Gandjbakhch; Alban Redheuil; Françoise Pousset; Philippe Charron; Robert Frank Journal: J Am Coll Cardiol Date: 2018-08-14 Impact factor: 24.094
Authors: Bin Zhou; Leah B Honor; Huamei He; Qing Ma; Jin-Hee Oh; Catherine Butterfield; Ruei-Zeng Lin; Juan M Melero-Martin; Elena Dolmatova; Heather S Duffy; Alexander von Gise; Pingzhu Zhou; Yong Wu Hu; Gang Wang; Bing Zhang; Lianchun Wang; Jennifer L Hall; Marsha A Moses; Francis X McGowan; William T Pu Journal: J Clin Invest Date: 2011-04-18 Impact factor: 14.808
Authors: Raffaella Lombardi; Maria da Graca Cabreira-Hansen; Achim Bell; Richard R Fromm; James T Willerson; A J Marian Journal: Circ Res Date: 2011-10-20 Impact factor: 17.367
Authors: Suet Nee Chen; Priyatansh Gurha; Raffaella Lombardi; Alessandra Ruggiero; James T Willerson; A J Marian Journal: Circ Res Date: 2013-11-25 Impact factor: 17.367
Authors: Ke Wei; Vahid Serpooshan; Cecilia Hurtado; Marta Diez-Cuñado; Mingming Zhao; Sonomi Maruyama; Wenhong Zhu; Giovanni Fajardo; Michela Noseda; Kazuto Nakamura; Xueying Tian; Qiaozhen Liu; Andrew Wang; Yuka Matsuura; Paul Bushway; Wenqing Cai; Alex Savchenko; Morteza Mahmoudi; Michael D Schneider; Maurice J B van den Hoff; Manish J Butte; Phillip C Yang; Kenneth Walsh; Bin Zhou; Daniel Bernstein; Mark Mercola; Pilar Ruiz-Lozano Journal: Nature Date: 2015-09-16 Impact factor: 49.962