Comparative study on transcriptomic differences between cardiomyocytes and cardiomyocyte nuclei

Abstract Background Global gene expression analysis is essential for understanding cellular functions in health and disease. In cardiac research, transcriptomic sequencing has progressed to the single-cell level. However, due to their large size, cardiomyocytes are not amenable to high-throughput scRNA-seq, making snRNA-seq the alternative. Given the distinct subcellular localization of RNA, spatial differences in gene expression may impact sequencing results. Understanding transcriptomic differences between nuclei and whole cardiomyocytes is crucial for accurate data interpretation. Purpose To compare the transcriptomic profiles of cardiomyocytes and their nuclei, offering insights for the interpretation and application of single-cell and single-nucleus transcriptomic data. Methods Cardiomyocytes and nuclei were isolated from adult male mice for bulk RNA sequencing. Neonatal cardiomyocytes and nuclei underwent scRNA-seq and snRNA-seq. Differential gene expression and functional enrichment analyses were performed, followed by experimental validation. Results Whole-cell transcripts exhibited a 3’ UTR preference, while nuclear splicing efficiency was significantly lower. Protein-coding genes showed similar expression in both nuclei and whole cells, while lncRNAs and snoRNAs were enriched in the nucleus. Differential expression analysis identified 4,217 upregulated and 3,255 downregulated genes in nuclei. GO enrichment analysis revealed that upregulated nuclear genes were involved in extracellular matrix organization and cell adhesion, whereas downregulated genes were associated with mitochondrial function and ATP metabolism. Analysis of nuclear-encoded mitochondrial protein (NEMP) genes showed higher transcript levels in whole cardiomyocytes. Both bulk RNA-seq and single-cell/single-nucleus RNA-seq confirmed that mitochondrial genes were more abundant in the cytoplasm, while cytoskeletal and extracellular matrix genes were enriched in the nucleus. Comparison of neonatal snRNA-seq and scRNA-seq data revealed differences in cardiomyopathy-related genes, but no clear localization preference. Notably, dilated cardiomyopathy GWAS genes and drug target genes were preferentially enriched in the nucleus. Conclusion This study provides a comprehensive resource on RNA subcellular localization in adult cardiomyocytes, revealing significant differences in mRNA and lncRNA distribution between whole cells and nuclei. We also offer new insights into the localization of nuclear-encoded mitochondrial transcripts, improving our understanding of gene regulation. Our results suggest that using nuclear RNA as a substitute for whole-cell RNA may introduce biases in gene expression analysis. These findings highlight the importance of considering subcellular localization when interpreting single-cell and single-nucleus sequencing data in cardiomyocytes.