High-throughput single-molecule RNA imaging analysis reveals heterogeneous responses of cardiomyocytes to hemodynamic overload

Abstract

A R T I C L E I N F O Keywords:Heart failure Single-cell spatial analysis Immunostaining Single-molecule fluorescence in situ hybridization A B S T R A C T Background: The heart responds to hemodynamic overload through cardiac hypertrophy and activation of the fetal gene program. However, these changes have not been thoroughly examined in individual cardiomyocytes, and the relation between cardiomyocyte size and fetal gene expression remains elusive. We established a method of high-throughput single-molecule RNA imaging analysis of in vivo cardiomyocytes and determined spatial and temporal changes during the development of heart failure. Methods and results: We applied three novel single-cell analysis methods, namely, single-cell quantitative PCR (sc-qPCR), single-cell RNA sequencing (scRNA-seq), and single-molecule fluorescence in situ hybridization (smFISH). Isolated cardiomyocytes and cross sections from pressure overloaded murine hearts after transverse aortic constriction (TAC) were analyzed at an early hypertrophy stage (2 weeks, TAC2W) and at a late heart failure stage (8 weeks, TAC8W). Expression of myosin heavy chain β (Myh7), a representative fetal gene, was induced in some cardiomyocytes in TAC2W hearts and in more cardiomyocytes in TAC8W hearts. Expression levels of Myh7 varied considerably among cardiomyocytes. Myh7-expressing cardiomyocytes were significantly more abundant in the middle layer, compared with the inner or outer layers of TAC2W hearts, while such spatial differences were not observed in TAC8W hearts. Expression levels of Myh7 were inversely correlated with cardiomyocyte size and expression levels of mitochondria-related genes. Conclusions: We developed a new image-analysis pipeline to allow automated and unbiased quantification of gene expression at the single-cell level and determined the spatial and temporal regulation of heterogenous Myh7 expression in cardiomyocytes after pressure overload.

Cardiac dopamine D1 receptor triggers ventricular arrhythmia in chronic heart failure

“…1c). We next examined the spatial expressions of Drd1 in the failing heart by using single-molecule fluorescence in situ hybridization (smFISH) with our previously developed algorithm, which allows us to assess mRNA levels of targeted genes in CMs 9 (Fig. 1c).…”

“…smFISH was performed with an RNAscope ® (Advanced Cell Diagnostics, Newark, CA) according to the manufacturer’s instruction 9 . The target probe against mouse Drd1a mRNA (NM_010076.3, bp 444-1358, ACD# 406491-C2) was designed using a previously described protocol 30 .…”

Single-cell RNA-sequencing Unravels Heterogeneity of Cardiomyocytes and Signaling Pathways in Pressure Overload

“…Increasing evidence suggests that it is of importance to probe out the mechanisms of cardiac hypertrophy by using scRNA-seq analysis. For instance, a previous study found that the abnormal expression of Myh7 in cardiomyocytes following pressure overload was regulated by time through scRNA-seq [22]. Another study reported that cardiac differentiation of human pluripotent stem cells could depend on HOPX based on scRNA-seq analysis [23].…”

Review of Single-Cell RNA Sequencing in the Heart

“…Some studies have investigated spatial gene expression in the heart. For example, Satoh et al spatially quantified gene expression at the single-cell level in the heart after TAC and found spatially heterogenous Myh7 expression in CMs after TAC [50]. Asp et al combined scRNA-seq data of human embryonic cardiac cells, RNA-seq data of spatial transcriptomics, and in situ sequencing data to generate a 3D gene expression atlas of the developing human heart [36].…”