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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.

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Embryonic cardiomyocytes co-express ITGA5 and ITGA6.E13.5 mouse hearts were manually dissociated and co-labeled with antibodies against ITGA6 and α-actinin (A) or ITGA5 and α-actinin (B). Histograms display surface marker expression of the upper right quadrant only. Surface marker segregate low (green) and high (red) expressing CMs. (C) FMO control, intracellular labeling of CMs without surface marker staining. “%” and “%-#” refer to the current gate, “%-2” refers to the parent gate.
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pone.0143538.g002: Embryonic cardiomyocytes co-express ITGA5 and ITGA6.E13.5 mouse hearts were manually dissociated and co-labeled with antibodies against ITGA6 and α-actinin (A) or ITGA5 and α-actinin (B). Histograms display surface marker expression of the upper right quadrant only. Surface marker segregate low (green) and high (red) expressing CMs. (C) FMO control, intracellular labeling of CMs without surface marker staining. “%” and “%-#” refer to the current gate, “%-2” refers to the parent gate.

Mentions: After the initial screening and validation experiments on chamber fractions, we next analyzed expression of ITGA5 and ITGA6 in whole heart preparations. As exemplary shown in Fig 2 virtually all E13.5 CMs expressed ITGA6 (Fig 2A) and ITGA5 (Fig 2B). ITGA6-expressing CMs segregated into a subpopulation with low fluorescence intensity (ITGA6low) and one with high fluorescence intensity (ITGA6high) (Fig 2A): 77.9% of CMs (24.4% of heart cells) were ITGA6low and 22.1% of CMs (6.9% of heart cells) were ITGA6high. Similar to ITGA6, ITGA5-positive CMs could be subdivided into two populations based on their fluorescence intensity. As depicted in Fig 2B, 64.6% of CMs (21.7% of heart cells) were ITGA5high and 35.4% of the CMs (11.9% of heart cells) were ITGA5low.


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)

Embryonic cardiomyocytes co-express ITGA5 and ITGA6.E13.5 mouse hearts were manually dissociated and co-labeled with antibodies against ITGA6 and α-actinin (A) or ITGA5 and α-actinin (B). Histograms display surface marker expression of the upper right quadrant only. Surface marker segregate low (green) and high (red) expressing CMs. (C) FMO control, intracellular labeling of CMs without surface marker staining. “%” and “%-#” refer to the current gate, “%-2” refers to the parent gate.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143538.g002: Embryonic cardiomyocytes co-express ITGA5 and ITGA6.E13.5 mouse hearts were manually dissociated and co-labeled with antibodies against ITGA6 and α-actinin (A) or ITGA5 and α-actinin (B). Histograms display surface marker expression of the upper right quadrant only. Surface marker segregate low (green) and high (red) expressing CMs. (C) FMO control, intracellular labeling of CMs without surface marker staining. “%” and “%-#” refer to the current gate, “%-2” refers to the parent gate.
Mentions: After the initial screening and validation experiments on chamber fractions, we next analyzed expression of ITGA5 and ITGA6 in whole heart preparations. As exemplary shown in Fig 2 virtually all E13.5 CMs expressed ITGA6 (Fig 2A) and ITGA5 (Fig 2B). ITGA6-expressing CMs segregated into a subpopulation with low fluorescence intensity (ITGA6low) and one with high fluorescence intensity (ITGA6high) (Fig 2A): 77.9% of CMs (24.4% of heart cells) were ITGA6low and 22.1% of CMs (6.9% of heart cells) were ITGA6high. Similar to ITGA6, ITGA5-positive CMs could be subdivided into two populations based on their fluorescence intensity. As depicted in Fig 2B, 64.6% of CMs (21.7% of heart cells) were ITGA5high and 35.4% of the CMs (11.9% of heart cells) were ITGA5low.

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