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Published on: August 20, 2014
High-throughput single-molecule RNA imaging analysis reveals heterogeneous responses of cardiomyocytes to hemodynamic
Masahiro Satoh1, Seitaro Nomura2, Mutsuo Harada3
1Department of Cardiovascular Medicine, Chiba University Graduate School of Medicine, Chiba, Japan; Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan.
Insights
Heart failure involves fetal gene activation in cardiomyocytes. This study reveals fetal gene expression inversely correlates with cardiomyocyte size and mitochondrial gene expression, offering new insights into heart disease progression.
Area of Science:
- Cardiovascular Biology
- Molecular Cardiology
- Systems Biology
Background:
- The heart adapts to hemodynamic overload via cardiac hypertrophy and fetal gene program activation.
- Individual cardiomyocyte responses and the link between cell size and fetal gene expression in heart failure are not well understood.
Purpose of the Study:
- To investigate spatial and temporal changes in gene expression within individual cardiomyocytes during heart failure development.
- To establish a high-throughput method for analyzing gene expression in vivo cardiomyocytes.
Main Methods:
- Utilized single-cell quantitative PCR (sc-qPCR), single-cell RNA sequencing (scRNA-seq), and single-molecule fluorescence in situ hybridization (smFISH).
- Analyzed cardiomyocytes from pressure-overloaded murine hearts (transverse aortic constriction) at early (2 weeks) and late (8 weeks) stages.
- Developed a novel image-analysis pipeline for automated, unbiased single-cell gene expression quantification.
Main Results:
- Myosin heavy chain β (Myh7) expression, a fetal gene marker, was induced in cardiomyocytes, increasing with heart failure progression.
- Myh7 expression varied significantly among cardiomyocytes and was inversely correlated with cardiomyocyte size and mitochondrial gene expression.
- Spatial differences in Myh7 expression were observed in early-stage hypertrophy, with higher abundance in the middle myocardial layer, which diminished in later stages.
Conclusions:
- Developed a new image-analysis pipeline for precise single-cell gene expression quantification in cardiomyocytes.
- Determined the spatial and temporal regulation of heterogeneous fetal gene expression in cardiomyocytes following pressure overload.
- Revealed an inverse relationship between fetal gene expression and cardiomyocyte size/mitochondrial activity during heart failure progression.
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.
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