Single-cell expression profiling reveals dynamic flux of cardiac stromal, vascular and immune cells in health and injury

Abstract

Besides cardiomyocytes (CM), the heart contains numerous interstitial cell types which play key roles in heart repair, regeneration and disease, including fibroblast, vascular and immune cells. However, a comprehensive understanding of this interactive cell community is lacking. We performed single-cell RNA-sequencing of the total non-CM fraction and enriched (Pdgfra-GFP+) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction (MI) surgery. Clustering of >30,000 single cells identified >30 populations representing nine cell lineages, including a previously undescribed fibroblast lineage trajectory present in both sham and MI hearts leading to a uniquely activated cell state defined in part by a strong anti-WNT transcriptome signature. We also uncovered novel myofibroblast subtypes expressing either pro-fibrotic or anti-fibrotic signatures. Our data highlight non-linear dynamics in myeloid and fibroblast lineages after cardiac injury, and provide an entry point for deeper analysis of cardiac homeostasis, inflammation, fibrosis, repair and regeneration.

High-resolution transcriptomic profiling of the heart during chronic stress reveals cellular drivers of cardiac fibrosis and hypertrophy

“…These include fibroblasts (Pdgfra, Col1a1), pericytes (Pdgfrb, Vtn), smooth muscle cells (SMCs; Acta2, Myh11), Schwann cells (Plp1, Kcna1), endothelial cells (Pecam, Ly6c1), macrophages (Fcgr1, Csf1r), and other immune cell populations 6 (granulocytes, B cells, T cells and NK cells). A subset of fibroblasts (Fibroblast-Wif1) showed distinct gene expression patterns including active Wnt signaling and specific expression of Wif1, in line with our previous report [4] and the findings of Farbehi et al [5]. Additional less well-characterized cell populations included CMs (Ttn, Myh6), epicardial cells (Msln, Upk3b), lymphatic endothelial cells (Lyve1, Cldn5) and endocardial cells (Npr3, Cytl1) ( Figure 1B-D).…”

“…Previous large-scale single cell transcriptomic studies exploring cardiac cellular diversity have failed to profile key cardiac cell populations including CMs [3][4][5][6] due to a reliance on dropletbased sequencing approaches that are incompatible with large cell isolation. Moreover, extraction of CMs from rodent hearts typically requires retro-aortic perfusion with proteases to liberate cells from the extracellular matrix; this is a relatively time-consuming process that has limited applicability for cell preparation in transcriptomic studies [7].…”

Single-cell RNA-seq analysis reveals the crucial role of Collagen Triplex Helix Repeat Containing 1 (CTHRC1) cardiac fibroblasts for ventricular remodeling after myocardial infarction

“…However, both populations of CF are characterized by different markers, most probably because of the low number of Mki67 + CF included in the scRNA-seq study, and the reporter mice used. Interestingly, Faberhi et al described 9 subpopulations of PDGFRα + CF with a similar transcriptomic profiles that our subpopulations of Col1α1-GFP + CF at 7dpi 30 . However, our data indicate that CF that express Pdgfrα are contained within Col1α1-GFP + CF ( Fig.…”

“…CTHRC1 is a secreted protein that has the ability to inhibit collagen matrix synthesis through inhibition of TGF-β signaling 20,21,33 and that appears as a top marker gene for IRCF. Although expression of Cthrc1 has been previously identified as a potential marker for activated CF after MI in mice 9,30 and humans 29 , the longer follow-up in our study (up to 30dpi) may explain why it was not previously identified as a key player in the cardiac repair process. According to our results, expression of Cthrc1 peaks at 14dpi and decreases thereafter.…”

“…Non-myocyte cardiac cell heterogeneity has been recently characterized based on FACS sorting different populations of immune, endothelial and cardiac fibroblast 14,28,29 or on the usage of lineage tracing models such as PDGFRα 30 or POSTN 9 reporter mice. Most of these studies, focus on the early phase after cardiac injury 30 and assessed other pathological conditions 9,28,29 .…”