Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Cytoskeleton-Associated Protein 4 as a New Modulator of Fibroblasts Activation
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- 9 July 2018
- journal article
- research article
- Published by Ovid Technologies (Wolters Kluwer Health) in Circulation
- Vol. 138 (2), 166-180
- https://doi.org/10.1161/CIRCULATIONAHA.117.030742
Abstract
Background: Genome-wide transcriptome analysis has greatly advanced our understanding of the regulatory networks underlying basic cardiac biology and mechanisms driving disease. However, so far, the resolution of studying gene expression patterns in the adult heart has been limited to the level of extracts from whole tissues. The use of tissue homogenates inherently causes the loss of any information on cellular origin or cell type-specific changes in gene expression. Recent developments in RNA amplification strategies provide a unique opportunity to use small amounts of input RNA for genome-wide sequencing of single cells. Methods: Here, we present a method to obtain high-quality RNA from digested cardiac tissue from adult mice for automated single-cell sequencing of both the healthy and diseased heart. Results: After optimization, we were able to perform single-cell sequencing on adult cardiac tissue under both homeostatic conditions and after ischemic injury. Clustering analysis based on differential gene expression unveiled known and novel markers of all main cardiac cell types. Based on differential gene expression, we could identify multiple subpopulations within a certain cell type. Furthermore, applying single-cell sequencing on both the healthy and injured heart indicated the presence of disease-specific cell subpopulations. As such, we identified cytoskeleton-associated protein 4 as a novel marker for activated fibroblasts that positively correlates with known myofibroblast markers in both mouse and human cardiac tissue. Cytoskeleton-associated protein 4 inhibition in activated fibroblasts treated with transforming growth factor triggered a greater increase in the expression of genes related to activated fibroblasts compared with control, suggesting a role of cytoskeleton-associated protein 4 in modulating fibroblast activation in the injured heart. Conclusions: Single-cell sequencing on both the healthy and diseased adult heart allows us to study transcriptomic differences between cardiac cells, as well as cell type-specific changes in gene expression during cardiac disease. This new approach provides a wealth of novel insights into molecular changes that underlie the cellular processes relevant for cardiac biology and pathophysiology. Applying this technology could lead to the discovery of new therapeutic targets relevant for heart disease.Keywords
This publication has 34 references indexed in Scilit:
- Inhibition of miR-15 Protects Against Cardiac Ischemic InjuryCirculation Research, 2012
- Primary culture of chick, mouse or human neural crest cellsNature Protocols, 2011
- Angiotensin II increases periostin expression via Ras/p38 MAPK/CREB and ERK1/2/TGF-β1 pathways in cardiac fibroblastsCardiovascular Research, 2011
- Molecular Pathways Underlying Cardiac Remodeling During Pathophysiological StimulationCirculation, 2010
- Revealing developmental networks by comparative transcriptomicsTranscription, 2010
- Fast and accurate short read alignment with Burrows–Wheeler transformBioinformatics, 2009
- Systematic and integrative analysis of large gene lists using DAVID bioinformatics resourcesNature Protocols, 2008
- Wnt-induced secreted protein-1 is a prohypertrophic and profibrotic growth factorAmerican Journal of Physiology-Heart and Circulatory Physiology, 2007
- Lipofuscin-induced autofluorescence of living neonatal rat cardiomyocytes in cultureMechanisms of Ageing and Development, 1994
- Collagenase- and trypsin-dissociated heart cells: A comparative ultrastructural studyJournal of Molecular and Cellular Cardiology, 1976