An autofluorescence-based method for the isolation of highly purified ventricular cardiomyocytes

Veronica Larcher; Paolo Kunderfranco; Marco Vacchiano; Pierluigi Carullo; Marco Erreni; Irene Salamon; Federico Simone Colombo; Enrico Lugli; Marta Mazzola; Achille Anselmo; Gianluigi Condorelli

doi:10.1093/cvr/cvx239

An autofluorescence-based method for the isolation of highly purified ventricular cardiomyocytes

Veronica Larcher, Paolo Kunderfranco, Marco Vacchiano, Pierluigi Carullo, Marco Erreni, Irene Salamon, Federico Simone Colombo, Enrico Lugli, Marta Mazzola, Achille Anselmo, Gianluigi Condorelli

Istituto Clinico Humanitas

Research output: Contribution to journal › Article › peer-review

Abstract

Aims: The aim of our study was to set up a simple and reliable isolation method of living ventricular cardiomyocytes (vCMs) for molecular and biological studies.

Methods: A standard technique for the retrograde perfusion of an enzymatic solution was used to isolate cardiac cells from adult mouse heart. Fluorescence-activated cell sorting (FACS) on adult murine cardiac ventricle cells was performed, comparing the intrinsic autofluorescence in the FITC channel and the forward scatter (FSC) parameter in order to isolate highly fluorescent cells. The expression of cell-specific mRNAs was assessed with real-time PCR in cells sorted on the basis of their FITC and FSC characteristics.

Results: We identified two distinct subpopulations of cells harvested after retrograde perfusion of wild-type heart: FITChigh/FSCdim and FITCdim/FSChigh. Immunophenotyping and mRNA analysis (qPCR and RNA sequencing) revealed that only FITChigh/FSCdim cells were highly enriched in CM markers. Genes with high expression in endothelial cells and fibroblasts were enriched in the FITCdim/FSChigh subpopulation. With the use of tdTomatofl/fl-α-myosin heavy chain MerCreMer+/-mouse heart, we found that tdTomato-positive vCMs were present in the FITChigh/FSCdim region but were only rare in the FITCdim/FSChigh fraction.

Conclusion: We have developed a simple and reliable method for the isolation of highly purified vCMs from the adult murine myocardium, avoiding fixation and permeabilization steps. These isolated vCMs can be used in particular for detailed molecular studies, avoiding contamination with other myocardial cell types.

Original language	English
Journal	Cardiovascular Research
DOIs	https://doi.org/10.1093/cvr/cvx239
Publication status	E-pub ahead of print - Dec 13 2017

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Journal Article

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10.1093/cvr/cvx239

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title = "An autofluorescence-based method for the isolation of highly purified ventricular cardiomyocytes",

abstract = "Aims: The aim of our study was to set up a simple and reliable isolation method of living ventricular cardiomyocytes (vCMs) for molecular and biological studies.Methods: A standard technique for the retrograde perfusion of an enzymatic solution was used to isolate cardiac cells from adult mouse heart. Fluorescence-activated cell sorting (FACS) on adult murine cardiac ventricle cells was performed, comparing the intrinsic autofluorescence in the FITC channel and the forward scatter (FSC) parameter in order to isolate highly fluorescent cells. The expression of cell-specific mRNAs was assessed with real-time PCR in cells sorted on the basis of their FITC and FSC characteristics.Results: We identified two distinct subpopulations of cells harvested after retrograde perfusion of wild-type heart: FITChigh/FSCdim and FITCdim/FSChigh. Immunophenotyping and mRNA analysis (qPCR and RNA sequencing) revealed that only FITChigh/FSCdim cells were highly enriched in CM markers. Genes with high expression in endothelial cells and fibroblasts were enriched in the FITCdim/FSChigh subpopulation. With the use of tdTomatofl/fl-α-myosin heavy chain MerCreMer+/-mouse heart, we found that tdTomato-positive vCMs were present in the FITChigh/FSCdim region but were only rare in the FITCdim/FSChigh fraction.Conclusion: We have developed a simple and reliable method for the isolation of highly purified vCMs from the adult murine myocardium, avoiding fixation and permeabilization steps. These isolated vCMs can be used in particular for detailed molecular studies, avoiding contamination with other myocardial cell types.",

keywords = "Journal Article",

author = "Veronica Larcher and Paolo Kunderfranco and Marco Vacchiano and Pierluigi Carullo and Marco Erreni and Irene Salamon and Colombo, {Federico Simone} and Enrico Lugli and Marta Mazzola and Achille Anselmo and Gianluigi Condorelli",

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month = dec,

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doi = "10.1093/cvr/cvx239",

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publisher = "Oxford University Press",

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T1 - An autofluorescence-based method for the isolation of highly purified ventricular cardiomyocytes

AU - Larcher, Veronica

AU - Kunderfranco, Paolo

AU - Vacchiano, Marco

AU - Carullo, Pierluigi

AU - Erreni, Marco

AU - Salamon, Irene

AU - Colombo, Federico Simone

AU - Lugli, Enrico

AU - Mazzola, Marta

AU - Anselmo, Achille

AU - Condorelli, Gianluigi

PY - 2017/12/13

Y1 - 2017/12/13

N2 - Aims: The aim of our study was to set up a simple and reliable isolation method of living ventricular cardiomyocytes (vCMs) for molecular and biological studies.Methods: A standard technique for the retrograde perfusion of an enzymatic solution was used to isolate cardiac cells from adult mouse heart. Fluorescence-activated cell sorting (FACS) on adult murine cardiac ventricle cells was performed, comparing the intrinsic autofluorescence in the FITC channel and the forward scatter (FSC) parameter in order to isolate highly fluorescent cells. The expression of cell-specific mRNAs was assessed with real-time PCR in cells sorted on the basis of their FITC and FSC characteristics.Results: We identified two distinct subpopulations of cells harvested after retrograde perfusion of wild-type heart: FITChigh/FSCdim and FITCdim/FSChigh. Immunophenotyping and mRNA analysis (qPCR and RNA sequencing) revealed that only FITChigh/FSCdim cells were highly enriched in CM markers. Genes with high expression in endothelial cells and fibroblasts were enriched in the FITCdim/FSChigh subpopulation. With the use of tdTomatofl/fl-α-myosin heavy chain MerCreMer+/-mouse heart, we found that tdTomato-positive vCMs were present in the FITChigh/FSCdim region but were only rare in the FITCdim/FSChigh fraction.Conclusion: We have developed a simple and reliable method for the isolation of highly purified vCMs from the adult murine myocardium, avoiding fixation and permeabilization steps. These isolated vCMs can be used in particular for detailed molecular studies, avoiding contamination with other myocardial cell types.

AB - Aims: The aim of our study was to set up a simple and reliable isolation method of living ventricular cardiomyocytes (vCMs) for molecular and biological studies.Methods: A standard technique for the retrograde perfusion of an enzymatic solution was used to isolate cardiac cells from adult mouse heart. Fluorescence-activated cell sorting (FACS) on adult murine cardiac ventricle cells was performed, comparing the intrinsic autofluorescence in the FITC channel and the forward scatter (FSC) parameter in order to isolate highly fluorescent cells. The expression of cell-specific mRNAs was assessed with real-time PCR in cells sorted on the basis of their FITC and FSC characteristics.Results: We identified two distinct subpopulations of cells harvested after retrograde perfusion of wild-type heart: FITChigh/FSCdim and FITCdim/FSChigh. Immunophenotyping and mRNA analysis (qPCR and RNA sequencing) revealed that only FITChigh/FSCdim cells were highly enriched in CM markers. Genes with high expression in endothelial cells and fibroblasts were enriched in the FITCdim/FSChigh subpopulation. With the use of tdTomatofl/fl-α-myosin heavy chain MerCreMer+/-mouse heart, we found that tdTomato-positive vCMs were present in the FITChigh/FSCdim region but were only rare in the FITCdim/FSChigh fraction.Conclusion: We have developed a simple and reliable method for the isolation of highly purified vCMs from the adult murine myocardium, avoiding fixation and permeabilization steps. These isolated vCMs can be used in particular for detailed molecular studies, avoiding contamination with other myocardial cell types.

KW - Journal Article

U2 - 10.1093/cvr/cvx239

DO - 10.1093/cvr/cvx239

M3 - Article

C2 - 29244142

SN - 0008-6363

JO - Cardiovascular Research

JF - Cardiovascular Research

ER -

An autofluorescence-based method for the isolation of highly purified ventricular cardiomyocytes

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