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Updated: Aug 3, 2026
Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm
Published on: October 4, 2018
Paired single-cell imaging of calcium and expression to map niches of identity and function
Alexander P Clark1, Payton Gergen1, Jeffrey J Saucerman1,2
1Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, United States of America.
Insights
We developed CARBONITE, a new imaging framework to link cardiomyocyte function and molecular identity. This method reveals that cell nucleation state, not just subtype markers, significantly impacts calcium dynamics in human iPSC-CMs.
Area of Science:
- Cardiology
- Cell Biology
- Biophysics
Background:
- Cellular identity is typically inferred from molecular markers, while function is measured separately, limiting single-cell resolution.
- In cardiomyocytes, this disconnect hinders understanding of functional heterogeneity in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).
- Existing methods lack the ability to directly link live functional measurements with molecular identity at the single-cell level.
Purpose of the Study:
- To introduce CARBONITE (Calcium Recordings Before Identification by Expression), a novel imaging framework.
- To enable quantitative functional-molecular mapping within the same cardiomyocyte.
- To investigate the relationship between calcium dynamics, molecular markers, and cell states in iPSC-CMs.
Main Methods:
- Developed CARBONITE, a scalable single-cell imaging framework integrating live calcium dynamics with protein expression and spatial phenotyping.
- Utilized high-content imaging in 96-well plates to pair per-cell calcium transient features with immunofluorescent identification.
- Applied the framework to mixed iPSC-CM populations to analyze functional and molecular heterogeneity.
Main Results:
- CARBONITE revealed significant heterogeneity in calcium transient dynamics and marker expression within iPSC-CM populations.
- Calcium transient shape segregated cells into two discrete functional states linked to perinuclear ANP enrichment and nucleation state (mono- vs. binucleation).
- Binucleated cells were more likely to exhibit spike-like calcium transients, indicating nucleation as a key factor influencing cardiomyocyte function.
Conclusions:
- CARBONITE is a functional multimodal single-cell platform for dissecting cell identity and function.
- Nucleation state is a dominant, underappreciated axis of cardiomyocyte identity influencing calcium handling.
- This platform provides a foundation for understanding functional niches in cardiac development and disease.
Abstract:
Cellular identity is often inferred from molecular markers, while function is measured independently, obscuring how these dimensions align at single-cell resolution. In cardiomyocytes, this disconnect is especially limiting, as calcium dynamics and subtype markers are typically assessed in bulk or separate cells then averaged across populations. In human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), this gap has limited our ability to determine whether heterogeneity in electrophysiology and calcium handling reflects noise, maturation, or structured biological states. This lack of clarity is due in part to the lack of methods that directly link live functional measurements with molecular identity at single-cell resolution. Here, we introduce CARBONITE (Calcium Recordings Before Identification by Expression), a scalable single-cell imaging framework that pairs live calcium dynamics with protein expression and spatial phenotyping. Using high-content imaging in 96-well plates, CARBONITE integrates per-cell calcium transient features with immunofluorescent identification applied to cardiomyocyte subtype markers, enabling quantitative functional-molecular mapping within the same cells. Applying CARBONITE to mixed human induced pluripotent stem cell derived cardiomyocyte (iPSC-CM) populations reveals heterogeneity in calcium transient dynamics and marker expression within individual wells. Canonical atrial and ventricular markers capture only a subset of functional variability. Notably, calcium transient shape segregates cells into two discrete functional states that exhibit perinuclear ANP (atrial marker) enrichment and nucleation state (i.e., mono- vs. binucleation). Notably, these groups show know relationship to MYL2 (ventricular marker) expression. Binucleated cells are more likely to exhibit a spike-like calcium transient, identifying nucleation as a dominant and previously underappreciated axis of cardiomyocyte identity influencing calcium function. Together, these results establish CARBONITE as a functional multimodal single-cell platform that reveals organizational principles of cell identity, providing a foundation for dissecting functional niches in development and disease.
