Pro-regenerative fingerprints identified in a sub-population of adult mouse cardiomyocytes by integrative single-cell proteomics.

Abstract

Recent advances in instrument sensitivity and sample preparation techniques are significantly improving the ability to study the heterogeneity of cell populations at the single-cell level by mass spectrometry-based proteomics. Integrating multiple layers of cellular data offers additional and yet unexplored insights in single-cell proteomics. In regenerative research, cardiomyocyte proliferation driven by the overexpression of the Myc transcription factor has been described, however, it has not yet been investigated at single-cell resolution. By using an optimized adult cardiomyocytes isolation procedure from mouse models and taking advantage of the integrative capabilities of the iSanXoT application, we are able to minimize batch effects and cell size-related biases, obtain quantitative subcellular compartment information and detect protein alterations within subcellular compartments, as it had not yet been defined for this methodology. This approach enhances data quantification accuracy and facilitates biological interpretation. We show that the Myc transcription factor switches the expression profile of metabolic enzymes and expands a subpopulation of adult cardiomyocytes with a pro-regenerative signature. We demonstrate that different layers of information can properly pattern the proteomic phenotype of single-cells. The analysis of single-cardiomyocyte data with the integrative statistical framework of iSanXoT provided important clues to understand the impact of Myc transcription factor in provoking different immaturity and pro-regenerative signatures in adult mouse cardiomyocytes. The cellular heterogeneity exerted by mouse cardiomyocytes upon Myc overexpression demonstrates the relevance of conducting regenerative studies at the single-cell level for precisely defining the amplitude of this response in the heart.