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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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Differential expression of ITGA5 and ITGA6 on atrial and ventricular cardiomyocytes.(A) E13.5 whole hearts and mechanically separated atrial and ventricular tissue were co-labeled for ITGA6 or ITGA5 and α-actinin. Histograms, ITGA6 or ITGA5 expression gated on α-actinin+ cells. (B) E13.5 whole-heart preparations co-stained with antibodies against ITGA6 or ITGA5 and MLC-2a or MLC-2v (labeled with AlexaFluor® 488 goat anti-mouse IgG). Analysis gates set according to the secondary antibody control. Rectangles indicate ITGA6 low (green) and high (red) expressing myocytes. (C) Co-labeling of E11.5 –P2 mouse hearts for ITGA6 and α-actinin.
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pone.0143538.g003: Differential expression of ITGA5 and ITGA6 on atrial and ventricular cardiomyocytes.(A) E13.5 whole hearts and mechanically separated atrial and ventricular tissue were co-labeled for ITGA6 or ITGA5 and α-actinin. Histograms, ITGA6 or ITGA5 expression gated on α-actinin+ cells. (B) E13.5 whole-heart preparations co-stained with antibodies against ITGA6 or ITGA5 and MLC-2a or MLC-2v (labeled with AlexaFluor® 488 goat anti-mouse IgG). Analysis gates set according to the secondary antibody control. Rectangles indicate ITGA6 low (green) and high (red) expressing myocytes. (C) Co-labeling of E11.5 –P2 mouse hearts for ITGA6 and α-actinin.

Mentions: When comparing the ITGA6 expression pattern in whole-hearts with atrial and ventricular preparations we detected two peaks for CMs in whole-heart preparations, but only one peak of high fluorescence intensity in the atrial and one of low intensity in the ventricular tissue fraction (Fig 3A). Regarding ITGA5 expression we found one tailing peak in whole-heart preparations, which could be split into one at low intensity for the atrial and one at high intensity for the ventricular preparation.


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)

Differential expression of ITGA5 and ITGA6 on atrial and ventricular cardiomyocytes.(A) E13.5 whole hearts and mechanically separated atrial and ventricular tissue were co-labeled for ITGA6 or ITGA5 and α-actinin. Histograms, ITGA6 or ITGA5 expression gated on α-actinin+ cells. (B) E13.5 whole-heart preparations co-stained with antibodies against ITGA6 or ITGA5 and MLC-2a or MLC-2v (labeled with AlexaFluor® 488 goat anti-mouse IgG). Analysis gates set according to the secondary antibody control. Rectangles indicate ITGA6 low (green) and high (red) expressing myocytes. (C) Co-labeling of E11.5 –P2 mouse hearts for ITGA6 and α-actinin.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4664422&req=5

pone.0143538.g003: Differential expression of ITGA5 and ITGA6 on atrial and ventricular cardiomyocytes.(A) E13.5 whole hearts and mechanically separated atrial and ventricular tissue were co-labeled for ITGA6 or ITGA5 and α-actinin. Histograms, ITGA6 or ITGA5 expression gated on α-actinin+ cells. (B) E13.5 whole-heart preparations co-stained with antibodies against ITGA6 or ITGA5 and MLC-2a or MLC-2v (labeled with AlexaFluor® 488 goat anti-mouse IgG). Analysis gates set according to the secondary antibody control. Rectangles indicate ITGA6 low (green) and high (red) expressing myocytes. (C) Co-labeling of E11.5 –P2 mouse hearts for ITGA6 and α-actinin.
Mentions: When comparing the ITGA6 expression pattern in whole-hearts with atrial and ventricular preparations we detected two peaks for CMs in whole-heart preparations, but only one peak of high fluorescence intensity in the atrial and one of low intensity in the ventricular tissue fraction (Fig 3A). Regarding ITGA5 expression we found one tailing peak in whole-heart preparations, which could be split into one at low intensity for the atrial and one at high intensity for the ventricular preparation.

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