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Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes.

Wiencierz AM, Kernbach M, Ecklebe J, Monnerat G, Tomiuk S, Raulf A, Christalla P, Malan D, Hesse M, Bosio A, Fleischmann BK, Eckardt D - PLoS ONE (2015)

Bottom Line: In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts.We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry.This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.

View Article: PubMed Central - PubMed

Affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.

ABSTRACT

Rationale: Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers.

Methods and results: In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis.

Conclusion: Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.

Show MeSH
Antibody-based surface marker screening of mechanically separated atrial and ventricular fraction (E13.5).(A) List of all markers that were expressed by a minimum of 10% of the cells from either atrial or ventricular cells. The expression frequency is color-coded. (B) Density plots, left panel, co-labeling of ALCAM and VCAM-1 on both fractions as resulted from the antibody screen. Right panel, validation of VCAM-1 as CM marker by co-labeling of VCAM-1 with cardiac troponin T. (C) Density plots, validation of differential expression of ITGA6 (left panel) and ITGA5 (right panel) by co-labeling with an antibody against cardiac troponin T.
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pone.0143538.g001: Antibody-based surface marker screening of mechanically separated atrial and ventricular fraction (E13.5).(A) List of all markers that were expressed by a minimum of 10% of the cells from either atrial or ventricular cells. The expression frequency is color-coded. (B) Density plots, left panel, co-labeling of ALCAM and VCAM-1 on both fractions as resulted from the antibody screen. Right panel, validation of VCAM-1 as CM marker by co-labeling of VCAM-1 with cardiac troponin T. (C) Density plots, validation of differential expression of ITGA6 (left panel) and ITGA5 (right panel) by co-labeling with an antibody against cardiac troponin T.

Mentions: From the candidates we first selected antibodies that labeled more than 10% of non-blood cells in at least one of the cardiac chamber fractions resulting in a list of 51 antibodies (Fig 1A). Next we chose antibodies either detecting co-expression with ALCAM, previously described as CM marker [6, 7, 8], or marking a strong difference of antigen expression between atria and ventricles. Validation experiments analyzed potential co-expression of cardiac troponin T and the respective surface marker candidates.


Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes.

Wiencierz AM, Kernbach M, Ecklebe J, Monnerat G, Tomiuk S, Raulf A, Christalla P, Malan D, Hesse M, Bosio A, Fleischmann BK, Eckardt D - PLoS ONE (2015)

Antibody-based surface marker screening of mechanically separated atrial and ventricular fraction (E13.5).(A) List of all markers that were expressed by a minimum of 10% of the cells from either atrial or ventricular cells. The expression frequency is color-coded. (B) Density plots, left panel, co-labeling of ALCAM and VCAM-1 on both fractions as resulted from the antibody screen. Right panel, validation of VCAM-1 as CM marker by co-labeling of VCAM-1 with cardiac troponin T. (C) Density plots, validation of differential expression of ITGA6 (left panel) and ITGA5 (right panel) by co-labeling with an antibody against cardiac troponin T.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664422&req=5

pone.0143538.g001: Antibody-based surface marker screening of mechanically separated atrial and ventricular fraction (E13.5).(A) List of all markers that were expressed by a minimum of 10% of the cells from either atrial or ventricular cells. The expression frequency is color-coded. (B) Density plots, left panel, co-labeling of ALCAM and VCAM-1 on both fractions as resulted from the antibody screen. Right panel, validation of VCAM-1 as CM marker by co-labeling of VCAM-1 with cardiac troponin T. (C) Density plots, validation of differential expression of ITGA6 (left panel) and ITGA5 (right panel) by co-labeling with an antibody against cardiac troponin T.
Mentions: From the candidates we first selected antibodies that labeled more than 10% of non-blood cells in at least one of the cardiac chamber fractions resulting in a list of 51 antibodies (Fig 1A). Next we chose antibodies either detecting co-expression with ALCAM, previously described as CM marker [6, 7, 8], or marking a strong difference of antigen expression between atria and ventricles. Validation experiments analyzed potential co-expression of cardiac troponin T and the respective surface marker candidates.

Bottom Line: In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts.We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry.This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.

View Article: PubMed Central - PubMed

Affiliation: Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.

ABSTRACT

Rationale: Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers.

Methods and results: In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis.

Conclusion: Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.

Show MeSH