Abstract 827: Single-cell Transcriptomic Profiling Provides Insights Into Disease-related Processes During Hypertrophic Cardiomyopathy

Abstract

Hypertrophic cardiomyopathy (HCM) is a genetic disease usually caused by a heterozygous mutation in a sarcomeric gene. Histologically, HCM is characterized by a heterogeneous population of hypertrophic, disorganized cardiomyocytes and fibrosis of heart tissue. This hypertrophic growth can lead to outflow tract obstruction which can ultimately lead to progressive heart failure due to cardiac overload. Here we use single-cell RNA sequencing (scRNA-Seq) on septal myectomy samples from patients suffering from HCM to interrogate cellular heterogeneity in disease context. Using our previously optimized digestion and sorting protocol we were able to obtain good quality RNA for single cell analysis. Bioinformatic clustering analysis of single cell transcriptomes revealed 8 transcriptomically distinct subpopulations of cardiomyocytes. Our data indicate an NPPA/NPPB enriched cluster of cells and show TTN to function as an important determinant of the cellular heterogeneity. Correlation analysis links TTN expression to the expression of, among others, cardiomyopathy-associated protein (CMYA) 5 and 3, while there appears to be a negative correlation with, among others, TMEM212. We were able to confirm these correlations by RT-PCR on bulk RNA in an additional cohort of 97 HCM myectomy samples. Additionally, immuno-histochemical staining on myectomy samples and explanted HCM hearts confirmed the transcriptional heterogeneity among cardiomyocytes. A unique advantage of FACS-based single cell sorting cardiomyocytes is the availability of index data for the sorted cells. Correlation analysis of forward scatter, as a proxy for cell size, confirmed both well-known stress markers MYL2 and MYL7 and additional genes to be upregulated in the larger cells. In summary, we show that FACS-based sorting of HCM cardiomyocytes allows for scRNA-Seq of intact cardiomyocytes. Initial analyses of the data could be validated in a wider cohort of comparable samples, showing reliability of the data. Finally, incorporation of index data reveals genes correlated with cell size. Further interrogation of this data has the potential to reveal novel insights into the pathogenesis of HCM.