Abstract 20023: An Autofluorescence-based Method for the Isolation Of Highly Purified Cardiac Ventriculocytes

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Introduction: Mice represent an important tool for modeling human diseases. In particular, adult murine cardiomyocytes are extensively used for cardiovascular and pathophysiological studies. The isolation of living ventricular cardiomyocytes (vCMs) for molecular and biological analyses has always been challenging.The aim of this study was to set up a simple and reliable method for isolating a pure population of live adult vCMs through fluorescence-activated cell sorting (FACS).

Methods and Results: We have developed a method for the straightforward isolation of highly purified vCMs from the other cell types present in the adult murine myocardium. FACS analysis on adult cardiac ventricle cells was performed comparing the intrinsic autofluorescence in the green channel and the Forward Scatter (FSC) parameter. We identified two distinct subpopulations in our samples: FITChigh/FSCdim and FITCdim/FSChigh (58.89%±2.49 and 30.23%±2.38, respectively). The immunophenotyping and mRNA analysis of both sorted subpopulations revealed that only FITChigh/FSCdim cells were highly enriched in CM markers. The method was validated using fluorescent tdTomatofl/fl-αMyosin Heavy Chain MerCreMer+/- cardiomyocytes. Data obtained showed tdTomato+ vCMs in the FITChigh/FSCdim region (89.36%±1.89), with very few events included in the FITCdim/FSChigh area (7.4%±1.97). Next we performed RNA sequencing to analyze global transcriptional profiles. Differential analysis revealed 1,644 less-expressed genes and 279 more-expressed genes in FITChigh/FSCdim versus FITCdim/FSChigh cells. The greater number of downregulated genes suggests that the FITChigh/FSCdim subpopulation is composed of cells with a common, unique signature and that the FITCdim/FSChigh subpopulation is composed of different cells types. Moreover, genes highly expressed in CMs were enriched in the FITChigh/FSCdim subset whereas genes with high expression in endothelial cells and fibroblasts were enriched in the other subpopulation.

Conclusions: In conclusion, the suggested methodological approach allows the isolation of live, pure vCMs without fixation and permeabilization steps. Thus, freshly isolated pure vCMs can be used for further specific molecular and biological studies.

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