Abstract 4147291: Examination of pre-mRNA from cardiomyocytes at single-cell resolution from post-infarcted mouse hearts

Background: RNA-seq provides a powerful tool to dissect cellular heterogeneity in diseased hearts. It generates reads from both mature RNA and pre-mRNA. Traditionally, only mature RNA transcripts are considered for analysis, but studying both species of transcripts from single-cell RNA-seq of cardiomyocytes in post-infarcted hearts can reveal novel insights into the dynamic transcriptional changes and regulatory mechanisms that occur during heart repair and regeneration Research question: Do nascent transcriptional events from pre-mRNA forecast the biological processes in failing hearts better than the mRNA and unravel the complexity of cardiomyocyte diversity? Aim: Execute an exon-intron analysis on cardiomyocyte single-cell RNAseq data obtained from post-infarcted mouse hearts Methods: Cardiomyocytes from mice (n=4) post-LAD ligation were isolated and single-cell RNAseq was performed using MegaKit v.2 (Parse Biosciences) on a NovaSeq 6000. Data was analyzed via the Parse pipeline and Seurat v5 . Pre-mRNA reference was built with AGAT . Gene set enrichment was done using fgsea . Sham mice without ligation (n=4) served as controls Results : We analyzed at least 70K cells for each transcript type and compared their enrichment profiles for post-infarcted hearts to sham. Infarction resulted in enrichment for biological processes predominantly for development and fatty acid metabolism, especially from pre-mRNA mapping (mRNA vs pre-mRNA; p= 4.2 x 10 -18 vs 4.8 x 10 -32 ). At the level of individual clusters, cardiomyocyte heterogeneity was revealed with cells enriched for distinct processes. Common to both types of transcripts were terms enriched for cell death (mRNA vs pre-mRNA; p=7.6 x 10 -3 vs 4.2 x 10 -3 ) , tissue remodeling ( p=4.3 x 10 -4 vs 9.6 x 10 -4 ) , and respiratory&metabolic activity ( p=8.9 x 10 -5 vs 7.2 x 10 -8 ). However, compared to mRNA, the pre-mRNA had more cell clusters enriched for terms related to increased protein production activity ( p = 7 x 10 -4 ), activation of key signaling pathways ( p =8.5 x 10 -4 ), and defense response ( p =2.1 x 10 -6 ). These additional processes show adaptive mechanisms that promisingly forecast cardiomyocyte repair and could be visualized by mapping pre-mRNA Conclusion: Examining pre-mRNA offers a realistic view of stressed cardiomyocytes’ transcriptional dynamics. This study could identify new biomarkers to predict the onset of heart failure. Further insights into transitioning cells could aid in developing therapies for regeneration