Single-cell transcriptomics reconstructs fate conversion from fibroblast to cardiomyocyte

Abstract

Summary Direct lineage conversion offers a new strategy for tissue regeneration and disease modeling. Despite recent success in directly reprogramming fibroblasts into various cell types, the precise changes that occur as fibroblasts progressively convert to target cell fates remain unclear. The inherent heterogeneity and asynchronous nature of the reprogramming process renders it difficult to study using bulk genomic techniques. Here, to overcome this limitation, we applied single-cell RNA-seq to analyze global transcriptome changes at early stages of induced cardiomyocyte (iCM) reprogramming1–4. Using unsupervised dimensionality reduction and clustering algorithms, we identified molecularly distinct subpopulations of cells along reprogramming. We also constructed routes of iCM formation, and delineated the relationship between cell proliferation and iCM induction. Further analysis of global gene expression changes during reprogramming revealed an unexpected down-regulation of factors involved in mRNA processing and splicing. Detailed functional analysis of the top candidate splicing factor Ptbp1 revealed that it is a critical barrier to the acquisition of CM-specific splicing patterns in fibroblasts. Concomitantly, Ptbp1 depletion promoted cardiac transcriptome acquisition and increased iCM reprogramming efficiency. Additional quantitative analysis of our dataset revealed a strong correlation between the expression of each reprogramming factor and the progress of individual cells through the reprogramming process, and led to the discovery of novel surface markers for enrichment of iCMs. In summary, our single cell transcriptomics approaches enabled us to reconstruct the reprogramming trajectory and to uncover heretofore unrecognized intermediate cell populations, gene pathways and regulators involved in iCM induction.

Unique Transcription Factor Functions Regulate Epigenetic and Transcriptional Dynamics During Cardiac Reprogramming

“…Epicardial cells were detected based on expression of genes such as Lrrn4 and Mgp (cluster 12, Figure 1E) (Xiao et al, 2018). Notably, the expression of these genes exclusively within endocardium and epicardium cells throughout our time course does not support a previous report that suggested these genes are expressed at early stages in reprogramming cells but subsequently repressed by GMT expression (Figure 1C-D) (Liu et al, 2017). Instead, our analysis suggests these distinct cell types persist in the population in low numbers and were not detected in the previous study due to a limitation in the number of cells captured.…”

“…Direct reprogramming of cardiac fibroblasts to cardiomyocytes has been achieved by ectopic expression of cardiac-enriched factors Ieda et al, 2010;Nam et al, 2013;Qian et al, 2012;Song et al, 2012). Ectopic expression of Gata4, Mef2c, and Tbx5 (GMT) is sufficient to alter the fibroblast epigenome and promote expression of genes associated with cardiomyocytes while simultaneously repressing the fibroblast gene program (Ieda et al, 2010;Liu et al, 2017;Zhou et al, 2016). Perturbation of epigenetic remodelers and ectopic expression of additional transcription factors such as Hand2 and MYOCD were found to influence reprogramming as well, with addition of Hand2 resulting in a greater portion of pacemaker-like cells Christoforou et al, 2013;Protze et al, 2012;Zhou et al, 2016).…”

Therapeutic approaches for cardiac regeneration and repair

“…Addition of ZNF281 to the reprogramming cocktail suppressed the expression of genes associated with the inflammatory response and modulated cardiac gene expression by interacting with the transcription factor GATA4 (REF 123 ). In other studies, suppressing the expression of genes encoding certain factors such as the Polycomb complex protein BMI1 and the splicing factor polypyrimidine tract-binding protein 1 (PTB) enhanced cardiac reprogramming, suggesting that these factors act as barriers to cardiac repro- gramming 124,125 . However, reaching a consensus protocol for in vitro direct reprogramming of fibroblasts into cardiomyocytes is difficult because in these studies the cardiomyocyte markers, source of fibroblasts, and the time of analysis differ.…”

EPRS Regulates Proline-rich Pro-fibrotic Protein Synthesis during Cardiac Fibrosis

“…Intriguingly, GMT (Gata4, Mef2c, Tbx5)-driven CF-to-CM trans-differentiation requires downregulation of almost completely overlapped members of ARSs 37 as found in CF-tomyofibroblast transition, including the same nine ARSs plus LARS, RARS, and KARS. The GO analysis in this report 37 has shown that ARS and ECM gene pathways are among the top enriched in a gene cluster that is unaltered in gene expression during CM-to-pre-iCM transition and drops during pre-iCM-to-iCM maturation. Among all altered ARSs, EPRS is dramatically reduced during this process, thereby possibly contributing to the silencing of collagen expression during CF-to-CM conversion.…”