Systematic dissection, preservation, and multiomics in whole human and bovine hearts
Abstract
Objectives
We sought to develop a rigorous, systematic protocol for the dissection and preservation of human hearts for biobanking that expands previous success in postmortem transcriptomics to multiomics from paired tissue.
Background
Existing cardiac biobanks consist largely of biopsy tissue or explanted hearts in select diseases and are insufficient for correlating whole organ phenotype with clinical data.
Methods
We demonstrate optimal conditions for multiomics interrogation (ribonucleic acid (RNA) sequencing, untargeted metabolomics) in hearts by evaluating the effect of technical variables (storage solution, temperature) and simulated postmortem interval (PMI) on RNA and metabolite stability. We used bovine (n=3) and human (n=2) hearts fixed in PAXgene or snap-frozen with liquid nitrogen.
Results
Using a paired Wald test, only two of the genes assessed were differentially expressed between left ventricular samples from bovine hearts stored in PAXgene at 0 and 12 hours PMI (FDR q<0.05). We obtained similar findings in human left ventricular samples, suggesting stability of RNA transcripts at PMIs up to 12 hours. Different library preparation methods (mRNA poly-A capture vs. rRNA depletion) resulted in similar quality metrics with both library preparations achieving >95% of reads properly aligning to the reference genomes across all PMIs for bovine and human hearts. PMI had no effect on RNA Integrity Number or quantity of RNA recovered at the time points evaluated. Of the metabolites identified (855 total) using untargeted metabolomics of human left ventricular tissue, 503 metabolites remained stable across PMIs (0, 4, 8, 12 hours). Most metabolic pathways retained several stable metabolites.
Conclusions
Our data demonstrate a technically rigorous, reproducible protocol that will enhance cardiac biobanking practices and facilitate novel insights into human CVD.