Cellular Fusion Drives Polyploidization In Human Cardiomyocytes

Polyploidization is the roadblock to cardiac regeneration. Although endoreplication has been considered to be the main source of polyploidization in cardiomyocytes, recent evidence indicates transient cardiomyocyte fusion and its potential role in cardiac development in zebrafish. To understand polyploidization, it is essential to trace the lineage of cardiomyocytes. The existing lineage method is not suitable for studying the lineage in human heart cardiomyocytes. However, the accumulation of clonal somatic DNA mutation can be traced, and the clonal structure of these somatic mutations can be used for lineage reconstruction of human cardiomyocytes as these non-inherited clonal somatic mutations record the unique history of each somatic cell originating from a zygote and can be used as “endogenous barcodes.” Our analysis of ultra-deep targeted sequencing of ≈500 single cardiomyocyte nuclei using a panel of 253 validated clonal sSNVs revealed that 75.7% of the sequenced cardiomyocytes could be classified into one of the nine clades categorized based on the presence or absence of these mutations. Interestingly, we found that cardiomyocytes can have mutations from more than one clade, indicating the possibilities of cellular fusion in cardiomyocytes. Our results suggest that at least ~10% of tetraploid cardiomyocytes are generated from the fusion mechanism during development, whereas 60% higher ploidy cardiomyocytes are generated from the fusion. Further, we performed ATAC Seq and transcriptome analysis to find out the types of cells that are fused to generate polyploid cardiomyocytes in the human heart. Analysis of the transcriptome and chromatin peaks of tetraploid and higher ploidy cells unraveled a population of cells (cluster 7) that expressed chromatin peaks for endothelial markers (EGFL7) as well as cardiomyocyte-specific markers like TBX5, PLN, etc. Our study demonstrates cellular fusion in the adult human heart and the cell types involved in this fusion event. Understanding the generation of polyploid cardiomyocytes in the human heart might enable us to advance the mechanistic understanding of cardiomyocyte regeneration with the long-term goal of stimulating therapeutic cardiac regeneration.