Transcriptional heterogeneity of fibroblasts is a hallmark of the aging heart

Abstract

Single-nucleus RNA-sequencing reveals cellular heterogeneity of cardiac cells in aging. To comprehensively decipher the expected cellular responses to intrinsic cardiac aging, we applied microdroplet-based single-nucleus RNA-sequencing (9) of cross-sections of snap-frozen heart samples from 3 syngeneic young male mice (12 weeks old) and 3 aged male mice (18 months old). In total, we analyzed 14,827 nuclei from young hearts and 12,981 nuclei from old hearts (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.131092DS1). Using t-distributed stochastic neighbor embedding (tSNE) (10), global dimension reduction was constructed from all samples to visualize clusters that were defined by cell-specific gene markers (Figure 1A and Supplemental Table 2). Alignment of samples indicated high reproducibility across samples (Supplemental Figure 1). Most of the cells were in G 1 phase, and no influence of aging on cell cycle activity was observed (Supplemental Figure 2). Unsupervised clustering revealed 15 distinct gene expression patterns (Figure 1A and Supplemental Figure 3). Using cell type-specific gene markers (Supplemental Table 2) and published mouse single-cell gene expression data (11, 12), 7 major cell types could be annotated, including fibroblasts (A, B), cardiomyocytes (A, B, C), endothelial cells (A, B, C), immune cells (A, B, C), pericytes, epicardial cells, and adipocytes (Figure 1A and Supplemental Figure 3). In particular, for fibroblasts, the unsupervised clustering revealed 2 main clusters, fibroblast A (79.42%) and fibroblast B (20.58%). Separation of these 2 clusters was not significant (Supplemental Figure 3B), and gene markers were very similar (Supplemental Table 2); moreover, these 2 clusters were almost equally populated by young and old cells. Analysis of the cell numbers in clusters of other cell types than fibroblasts showed in part trends for changes during aging (Supplemental Figure 4) but did not reveal statistically significant differences. In general, 128 differentially expressed nonredundant genes (DEGs) were found between young and aged hearts (Figure 1B and Supplemental Table 3). Considering the DEGs in all cell clusters, 107 genes showed significantly increased expression (adjusted P < 0.1), and 21 genes showed significantly decreased expression (adjusted P < 0.1) in aged versus young hearts (Supplemental Table 3). Interestingly, aging predominantly affected gene expression patterns in fibroblasts (Figure 1B). Several highly differentially expressed genes could be confirmed by quantitative reverse transcription PCR of isolated cardiac fibroblasts (Supplemental Figure 5). Gene Ontology (GO) analysis of DEGs revealed a cell type-specific enrichment of genes associated with various pathways, such as angiogenesis, chemotaxis/migration, inflammation/immune response, and cell/matrix association (Figure 1C). Only a few coexpression networks and significantly regulated genes were shared between the main cell types. Among them, the e...

Single-nucleus RNA-seq identifies transcriptional heterogeneity in multinucleated skeletal myofibers

“…In addition to functional diversity found in normal development, transcriptional heterogeneity can arise from pathological or compensatory processes associated with aging [31][32][33] . Skeletal muscle undergoes dramatic age-related changes with respect to functional deterioration and regenerative decline [34][35][36][37] , and so we next asked if the aging process impacted myonuclear transcription.…”

Cellular cross-talks in the diseased and aging heart

“…Interestingly, this FB subset was shown to be reduced after MI [7]. While these examples highlight the interaction of FBs with CM, bioinformatics analysis of single cell sequencing data sets suggest that the main interacting cell type of fibroblasts in the heart may be endothelial cells, which may trigger further research to elucidate the interactions between these two cell types [7,89].…”

“…Single nucleus sequencing revealed that cardiac FBs of the aging heart highly express the SASP factor PAI-1. PAI-1 was further identified as one of the crucial factors in conditioned medium from age-heart derived FB, which interferes with endothelial cell angiogenesis [89]. In addition, mesenchymal stromal cells display senescence and SASP in the aging heart, which contributes to the recruitment of CCR-2-dependent monocytes.…”

Review of Single-Cell RNA Sequencing in the Heart

“…Intact adult CMs are too large to apply to a droplet system, and thus some researchers have extracted CM nuclei for use with this system to perform high-throughput scRNA-seq. Single-nucleus RNA-seq (snRNA-seq) of tens of thousands of nuclei extracted from healthy mice and from a murine MI model were performed using Chromium [23,24]. The results revealed that dedifferentiation may be an important prerequisite for CM proliferation and demonstrated the limited but measurable cardiac myogenesis after MI.…”

“…In the heart, ligand-receptor analysis revealed interactions between cardiac cell types in homeostasis and injury [23,27]. In particular, there is a strong association between fibroblasts and endothelial cells after injury or during aging.…”