Figure 4. scRNA-seq analyses predict mechanisms of polyploidization.
(A) Representative FACS plot of CCNB1-eGFP CMs stained with Hoechst to collect 2n, 4n CCNB1+, and 4n CCNB1− single-CMs in wells of 384-well plates.
(B) UMAP clustering analysis by Seurat uncovers seven CM clusters.
(C–E) Segregation of Seurat clusters by (C) CCNB1 and ploidy status identifies clusters abundant for 4n CCNB1− (0 and 1), 2n (3 and 5), and 4n CCNB1+ CMs (4 and 6), in accord with (D) CCNB1 transcript levels and (E) proportional breakdown of Seurat clusters by sorted cell type.
(F) Gene set enrichment analysis (GSEA) using the top 100 significantly upregulated genes (adjusted p ≤ 0.05) from clusters 4 and 6 (containing ~94% CCNB1+ CMs) identified transcripts related to the cell cycle, mitosis, and cell division.
(G) GSEA using the top 100 significantly upregulated genes from clusters 0 and 1 (containing ~66% of polyploid CMs) identified transcripts related to sarcomere function and structure, oxidative metabolism, and p53 signaling.
(H) Single CMs are displayed using UMAP overlapped with Slingshot pseudotime trajectories. Trajectory 1 begins in the diploid-abundant cluster 5 and ends in the CCNB1-abundant cluster 4.
(I and J) The top 100 genes defining trajectory 1 have (I) functions related to mitosis and the cell cycle, such as (J) G2/M checkpoint (CDK1 and UBE2C) and proliferation markers (MKI67).
(K–M) Trajectory 2 (K) begins in the diploid-abundant cluster 5 and ends in the polyploid-abundant cluster 0 and is (L) defined by genes related to sarcomere function and structure, oxidation, and p53 activity, such as (M) MYH6, TPM1, and CDKN1A, suggesting a causal relationship with polyploidization.