Single cell transcriptomic analyses of human heart failure with preserved ejection fraction

Abstract

BackgroundHeart failure with preserved ejection fraction (HFpEF) is a poorly understood, multi-system disease with high morbidity and mortality. To improve our understanding of its underlying biology, we used single-nucleus RNA sequencing (snRNA-seq) to characterize cell-specific gene expression patterns in human HFpEF myocardium. MethodsSeptal myocardial biopsies (2-3 mg) from 30 HFpEF patients and 29 non-failing donor controls were analyzed using the 10X Genomics platform, with nuclei isolated from combined samples (6 patients/pool). Genotype-based demultiplexing was performed with souporcell, and gene expression quantified with CellRanger and CellBender. After quality control, nuclei were clustered and annotated by cell types based on specific marker genes. Differential expression (DE) by cell-type in HFpEF vs controls was performed using limma-voom and functional analysis performed using Gene Set Enrichment Analysis. Data were compared to dilated cardiomyopathy (DCM) using prior snRNA-seq in DCM vs respective controls. ResultsWe successfully demultiplexed pooled myocardial biopsies, assigning >75% of droplets to individual patients. From eight pooled samples (19 HFpEF, 24 controls), we recovered 48,886 nuclei and identified 14 cell types. Cardiomyocytes (5159 differentially expressed [DE] genes, 36%) and fibroblasts (5905 DE genes, 49%) showed the most DE genes, while endothelial cells (2143), pericytes (1812), and macrophages (1405) had fewer. Enriched pathways common to multiple cell types included transcription/translation, immune activation, metabolism, and protein quality control. Of 7848 DE genes identified via pseudo-bulk snRNA-seq, 51% were DE in fibroblasts and 47% in cardiomyocytes, compared to <20% in other cell types. Unlike dilated cardiomyopathy (DCM), sub-clustering fibroblasts did not reveal an activated fibroblast population in HFpEF. Comparative analysis between HFpEF and DCM identified transcriptional differences primarily in cardiomyocytes. ConclusionsThis study demonstrates the power of genotype-based demultiplexing for single-cell transcriptomic analyses of small endomyocardial biopsies and identifies cardiomyocytes as the principal cell type with distinct transcriptional changes in HFpEF versus DCM. These findings, coupled with differential gene expression and functional pathway analyses, illuminate HFpEF pathways and may nominate compelling targets for future mechanistic studies and therapeutic efforts for HFpEF. Clinical PerspectiveWhat is new? We successfully used genotype-based demultiplexing to perform single nucleus RNA-seq from myocardial biopsies. The snRNA-seq analysis revealed distinct enrichment of pathways related to transcription/translation, immune activation, metabolism, and protein quality control across multiple cell types in HFpEF. In contrast to DCM, HFpEF is distinguished by the absence of an activated fibroblast population and a predominance of transcriptional differences within cardiomyocytes, highlighting a distinct disease mechanism. What are the clinical implications? The cell-selective HFpEF myocardial transcriptional landscape highlights altered metabolism, protein translation and quality control, immune activation and growth/matrix organization. These pathways provide compelling new targets for developing effective treatments.