Single-Cell Splicing Isoform Atlas of the Adult Human Heart and Heart Failure

BACKGROUND: Alternative splicing plays crucial roles in normal heart development and cardiac disease by influencing protein-coding sequences, functional domains, and molecular networks. However, a detailed characterization of the human heart isoform landscape remains incomplete. METHODS: Leveraging long-read single-nucleus RNA sequencing and computational analysis, we dissected full-length isoform heterogeneities, expression patterns, and usage shifts across cell types, cell states, and cardiac conditions of the adult left ventricle. We applied in silico approaches to assess the functional relevance of identified isoforms; validated isoform compositions of representative cardiac genes using reverse transcription quantitative polymerase chain reaction and targeted amplicon sequencing; and developed a web server for interactive navigation of our results. RESULTS: The data revealed that isoform heterogeneity is widespread in the cardiac cellular system, serving as a posttranscriptional buffer system that calibrates the molecule reservoirs in human hearts. In healthy left ventricles, ˜30% of cell type–specific genes were polyform, using multiple isoforms tailored to cell type–specific programs. Among ubiquitously expressed genes, >300 showed differential isoform usage with cell type specificity. Compared with heart failure, 379 genes in cardiomyocytes demonstrated marked isoform usage shifts, most of which are predicted to change protein coding outcomes through direct changes in protein coding sequences and switches between intron retention and non–protein-coding biotypes. In contrast, cell state–specific programs tend to operate on monoform genes associated with changes among cell states. In addition, our data revealed heart failure–associated differential isoform usage events in stromal and immune cell types in the cardiac microenvironment. CONCLUSIONS: We present a comprehensive atlas of splicing isoforms in the normal adult heart and heart failure through long-read single-nucleus RNA sequencing and comprehensive computational analyses. The results suggest crucial roles of isoforms in buffering core cellular programs and contributing to disease-associated cell states. The full-length details of these cell-specific isoforms serve as an important reference for downstream translational and mechanistic studies and are available on our online data portal at https://github.com/gaolabtools/heart-isoform-atlas. Github repo: https://github.com/gaolabtools/heart-isoform-atlas doi: 10.1161/CIRCULATIONAHA.125.074959 Overall design: Single-nucleus Nanopore RNA-sequencing of 12 frozen human left ventricle samples (6 non-diseased, 6 ischemic cardiomyopathy)