Northwestern Medicine scientists have developed a comprehensive atlas of genetic coding sequences in both healthy adult hearts and those with heart failure, a resource that has the potential to improve the understanding of heart health and inform the development of new therapeutic targets for cardiovascular disease, as detailed in a recent study published in Circulation.
“Our study offers a valuable resource of splicing isoforms in both the non-diseased and diseased left ventricle of adult human hearts, contributing to a deeper understanding of their roles in cardiac health and pathogenesis. The full-length details of these cell-specific isoforms serve as a critical reference for downstream translational and mechanistic studies,” said Ruli Gao, PhD, assistant professor of Biochemistry and Molecular Genetics and senior author of the study.
Alternative RNA splicing plays a key role in cardiac development and the manifestation of cardiovascular disease by operating on mRNAs that produce different coding sequences, or RNA isoforms, from the same gene.
“In normal heart development, alternative splicing allows for temporal, spatial and tissue-specific regulation of protein diversity, supporting various cardiac functions in development. In heart diseases, abnormal splicing regulation can affect key cardiac contraction and electrical conduction through mis-spliced mRNAs and their protein products, contributing to diseases such as cardiomyopathy, arrhythmias and heart failure,” said Gao, who is also a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University.
Previously, Gao and her team developed a novel high throughput long-read single-nucleus RNA-sequencing method that can profile thousands of full-length transcripts in individual cells from frozen cardiac tissue samples.
Using their approach coupled with computational analyses, the investigators developed a comprehensive atlas of splicing isoforms in healthy adult heart tissue as well as cardiac tissue with heart failure at cell-type resolution.
First, the scientists applied their long-read single-nucleus approach to left ventricle tissue samples from healthy adult hearts and those with heart failure. Next, they used computational analyses to dissect full-length isoforms from multiple aspects, such as isoform heterogeneities, expression patterns and usage shifts across cell types, cell states and cardiac conditions. Lastly, they applied in silico approaches to assess the functional relevance of identified isoforms and experimentally validated the accuracy of their approaches using top isoforms.
Their analyses revealed that isoform heterogeneity is widespread across cell types and cell states in the cardiac cellular system.
For example, in healthy left ventricles, approximately 30 percent of cell type-specific expressed genes are “polyform,” meaning they utilize multiple isoforms tailored to buffer and maintain core cellular programs. Over 300 ubiquitously expressed genes in hearts, such as TNNI3 and ACTG1, are associated with cell type-specific programs through differential isoform usage. When compared with heart failure, a total of 379 genes in cardiomyocytes demonstrates marked isoform usage shifts, including FRY, an evolutionarily conserved microtubule gene.
“Noteworthy, most of these genes involved in isoform usage shift were missed by traditional gene expression analyses,” said Timothy Pan, a student in the Driskill Graduate Program in Life Sciences (DGP), who was the lead author of the study.
The results emphasize the critical role of RNA isoforms in supporting key cellular programs and contributing to disease-associated cell states, according to Gao.
“Long-read sequencing of cell type-specific isoforms in human hearts under real pathophysiological conditions allows for the precise and comprehensive investigation of coding sequences of transcripts relevant to both normal and disease status,” Gao said. “While RNA isoforms are essential in understanding cardiac diseases, the full-length details of transcript sequences revealed by this study enable better discovery of new therapeutic targets and the development of more accurate diagnostic strategies.”
Co-authors of the study include Lina Lu, PhD, Cheng-Kai Shiau, PhD, and Minhua Wang, PhD, postdoctoral fellows in the Gao laboratory; Eric Tong, a DGP student; Arjun Sinha, MD, ‘21 MS, clinical assistant professor of Medicine in the Division of Cardiology; Ankit Bharat, MBBS, the Harold L. and Margaret N. Method Professor of Surgery; and Jane Wilcox, MD, ‘15 MSc, associate professor of Medicine in the Division of Cardiology.
This work was supported by Northwestern University, the National Heart, Lung, and Blood Institute (1R01HL160552) and National Institute of General Medical Sciences (R35GM142539).
