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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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Related in: MedlinePlus

Selective enrichment of neonatal atrial and ventricular cardiomyocytes.(A) P2 hearts were dissociated, depleted from cardiac non-myocytes and then sorted into ITGA6low (PL) and ITGA6high (PH) cells. (B) Density plots, distribution of cells before and after sorting. Dot plots, staining of the fractions for α-actinin and MLC-2a (labeled with APC anti-mouse IgG1 or IgG2ab). (C) Statistical analysis of α-actinin and MLC-2a before and after sorting. Data are expressed as mean ± SD, n = 3. t-test for paired samples with ** p ≤ 0.01 vs. before sorting.
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pone.0143538.g005: Selective enrichment of neonatal atrial and ventricular cardiomyocytes.(A) P2 hearts were dissociated, depleted from cardiac non-myocytes and then sorted into ITGA6low (PL) and ITGA6high (PH) cells. (B) Density plots, distribution of cells before and after sorting. Dot plots, staining of the fractions for α-actinin and MLC-2a (labeled with APC anti-mouse IgG1 or IgG2ab). (C) Statistical analysis of α-actinin and MLC-2a before and after sorting. Data are expressed as mean ± SD, n = 3. t-test for paired samples with ** p ≤ 0.01 vs. before sorting.

Mentions: As ALCAM, VCAM-1 as well as ERBB-2 expression decreased after birth (S3 Fig), the two-marker sorting strategy was restricted to embryonic hearts. Therefore, neonatal CMs were first purified using magnetic cell sorting (Fig 5A), and second, were separated into CM subtypes based on ITGA6 expression intensities, i.e. ITGA6low (PL) and ITGA6high cells (PH). As expected from the previous experiments, the two fractions highly differed with regard to MLC-2a expression: 70% MLC-2a+ cells in PH, 4% MLC-2a+ in PL (Fig 4B). Altogether, both isolation strategies repeatedly resulted in a highly selective enrichment or depletion of MLC-2a+ cells from mouse hearts (Figs 4E and 5C).


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)

Selective enrichment of neonatal atrial and ventricular cardiomyocytes.(A) P2 hearts were dissociated, depleted from cardiac non-myocytes and then sorted into ITGA6low (PL) and ITGA6high (PH) cells. (B) Density plots, distribution of cells before and after sorting. Dot plots, staining of the fractions for α-actinin and MLC-2a (labeled with APC anti-mouse IgG1 or IgG2ab). (C) Statistical analysis of α-actinin and MLC-2a before and after sorting. Data are expressed as mean ± SD, n = 3. t-test for paired samples with ** p ≤ 0.01 vs. before sorting.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143538.g005: Selective enrichment of neonatal atrial and ventricular cardiomyocytes.(A) P2 hearts were dissociated, depleted from cardiac non-myocytes and then sorted into ITGA6low (PL) and ITGA6high (PH) cells. (B) Density plots, distribution of cells before and after sorting. Dot plots, staining of the fractions for α-actinin and MLC-2a (labeled with APC anti-mouse IgG1 or IgG2ab). (C) Statistical analysis of α-actinin and MLC-2a before and after sorting. Data are expressed as mean ± SD, n = 3. t-test for paired samples with ** p ≤ 0.01 vs. before sorting.
Mentions: As ALCAM, VCAM-1 as well as ERBB-2 expression decreased after birth (S3 Fig), the two-marker sorting strategy was restricted to embryonic hearts. Therefore, neonatal CMs were first purified using magnetic cell sorting (Fig 5A), and second, were separated into CM subtypes based on ITGA6 expression intensities, i.e. ITGA6low (PL) and ITGA6high cells (PH). As expected from the previous experiments, the two fractions highly differed with regard to MLC-2a expression: 70% MLC-2a+ cells in PH, 4% MLC-2a+ in PL (Fig 4B). Altogether, both isolation strategies repeatedly resulted in a highly selective enrichment or depletion of MLC-2a+ cells from mouse hearts (Figs 4E and 5C).

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
Related in: MedlinePlus