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Canadian Journal of Cardiology

PROTOCOL DEVELOPMENT FOR SINGLE-NUCLEUS RNA SEQUENCING OF HUMAN HEART TISSUE

      Background

      Heart disease is a leading cause of global morbidity and mortality. This is partly because despite an abundance of animal and in-vitro models, it has been challenging to study human heart tissue with sufficient depth and resolution to develop disease modifying therapies for common cardiac conditions. Single-nucleus RNA sequencing (snRNAseq) has emerged as a powerful tool capable of analyzing cellular function and signalling in health and disease. Employing snRNAseq to study the human heart has the potential to unlock novel disease mechanisms and pathways. However, progress on this front has been slowed by several barriers. One key challenge is the fact that human heart tissue is very resistant to shearing and stress, making tissue dissociation and nuclear isolation difficult. Here, we describe a tissue dissociation method allowing for efficient and cost-effective isolation of high-quality nuclei from flash frozen human heart tissue collected in surgical operating rooms.

      Methods and Results

      Flash frozen human cardiac tissue was obtained through an established cardiac tissue biobank. Human cardiac tissue was collected and flash frozen from operating rooms performing open heart surgery. A protocol to generate single nuclei preparations from human tissue samples was found through literature review. This protocol was trialed and adapted for flash frozen human cardiac tissue. Assessments of protocol efficacy were made via nuclear morphology on brightfield imaging, DAPI stained nuclear morphology on epi-fluorescence imaging, Tapestation analysis of RNA integrity and concentration, and snRNAseq of isolated single nuclei to assess for cell type variability. Preliminary snRNAseq results are presented. Our results show that this modified protocol can reliably dissociate human cardiac tissue and isolate a high concentration of single nuclei. The preliminary snRNAseq results of isolated single nuclei using this protocol demonstrate high cell type variability.

      Conclusion

      Our protocol addresses the challenge of nuclear isolation from frozen human hearts and allows for snRNAseq of the human heart. Preliminary snRNAseq results show that this protocol can isolate high concentrations of single nuclei and from a diverse array of cell types. This suggests that sequencing findings from nuclei isolated using this protocol may be more representative of the in-vivo cellular composition of the human heart. This paves the way for an improved understanding of the human heart in health and disease. Ultimately, this will be key to uncovering signalling pathways and networks amenable to therapeutic intervention and the development of novel biomarkers and disease-modifying therapies.