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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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Gene expression analysis of sorted cells confirms selective enrichment of atrial and ventricular cardiomyocytes.(A) Normalized signal intensities of CM-specific marker genes: general CM-specific Tnnt2 and Nkx2-5, ventricle-specific Hey2 and Irx4, atrium-specific Nr2f2 and Fgf12. Data are expressed as mean ± SD, n = 4. Statistical analysis: ANOVA, Benjamini-Hochberg correction for multiple testing p ≤ 0.05, Tukey post-hoc test *** p ≤ 0.001, ns = not significant. (B) Heat-map shows median-centered log2-transformed signal intensities of selected genes. The color code indicates expression relative to the gene-wise median of all samples. Abbreviation: EL = E15.5 ERBB-2+/ITGA6low, EH = E15.5 ERBB-2+/ITGA6high, PL = P2 ITGA6low, PH = P2 ITGA6high.
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pone.0143538.g006: Gene expression analysis of sorted cells confirms selective enrichment of atrial and ventricular cardiomyocytes.(A) Normalized signal intensities of CM-specific marker genes: general CM-specific Tnnt2 and Nkx2-5, ventricle-specific Hey2 and Irx4, atrium-specific Nr2f2 and Fgf12. Data are expressed as mean ± SD, n = 4. Statistical analysis: ANOVA, Benjamini-Hochberg correction for multiple testing p ≤ 0.05, Tukey post-hoc test *** p ≤ 0.001, ns = not significant. (B) Heat-map shows median-centered log2-transformed signal intensities of selected genes. The color code indicates expression relative to the gene-wise median of all samples. Abbreviation: EL = E15.5 ERBB-2+/ITGA6low, EH = E15.5 ERBB-2+/ITGA6high, PL = P2 ITGA6low, PH = P2 ITGA6high.

Mentions: To further characterize the subpopulations, cells were sorted from E15.5 and P2 mouse hearts in quadruplicates and subjected to microarray analysis. A correlation analysis of the complete dataset resulted in a clear separation of the different sample groups (S4 Fig). The normalized signal intensities for general CM markers such as cardiac troponin T (Tnnt2) and NK2 homeobox 5 (Nkx2-5) were high and did not significantly vary between ITGA6low (EL, PL) and ITGA6high cells (EH, PH) (Fig 6A). However, several genes were restricted to one of the sorted fractions. For example, ventricle-specific transcripts Hey2 or Irx4 could be detected in EL and PL, but not or only at very low levels in EH and PH cells. In contrast, several genes associated with atrial myocytes were found to be weakly or not at all expressed in the EL and PL fractions, but were highly expressed in the EH and PH fractions, among them Fgf12 and Nr2f2 (encoding COUP-TFII).


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)

Gene expression analysis of sorted cells confirms selective enrichment of atrial and ventricular cardiomyocytes.(A) Normalized signal intensities of CM-specific marker genes: general CM-specific Tnnt2 and Nkx2-5, ventricle-specific Hey2 and Irx4, atrium-specific Nr2f2 and Fgf12. Data are expressed as mean ± SD, n = 4. Statistical analysis: ANOVA, Benjamini-Hochberg correction for multiple testing p ≤ 0.05, Tukey post-hoc test *** p ≤ 0.001, ns = not significant. (B) Heat-map shows median-centered log2-transformed signal intensities of selected genes. The color code indicates expression relative to the gene-wise median of all samples. Abbreviation: EL = E15.5 ERBB-2+/ITGA6low, EH = E15.5 ERBB-2+/ITGA6high, PL = P2 ITGA6low, PH = P2 ITGA6high.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664422&req=5

pone.0143538.g006: Gene expression analysis of sorted cells confirms selective enrichment of atrial and ventricular cardiomyocytes.(A) Normalized signal intensities of CM-specific marker genes: general CM-specific Tnnt2 and Nkx2-5, ventricle-specific Hey2 and Irx4, atrium-specific Nr2f2 and Fgf12. Data are expressed as mean ± SD, n = 4. Statistical analysis: ANOVA, Benjamini-Hochberg correction for multiple testing p ≤ 0.05, Tukey post-hoc test *** p ≤ 0.001, ns = not significant. (B) Heat-map shows median-centered log2-transformed signal intensities of selected genes. The color code indicates expression relative to the gene-wise median of all samples. Abbreviation: EL = E15.5 ERBB-2+/ITGA6low, EH = E15.5 ERBB-2+/ITGA6high, PL = P2 ITGA6low, PH = P2 ITGA6high.
Mentions: To further characterize the subpopulations, cells were sorted from E15.5 and P2 mouse hearts in quadruplicates and subjected to microarray analysis. A correlation analysis of the complete dataset resulted in a clear separation of the different sample groups (S4 Fig). The normalized signal intensities for general CM markers such as cardiac troponin T (Tnnt2) and NK2 homeobox 5 (Nkx2-5) were high and did not significantly vary between ITGA6low (EL, PL) and ITGA6high cells (EH, PH) (Fig 6A). However, several genes were restricted to one of the sorted fractions. For example, ventricle-specific transcripts Hey2 or Irx4 could be detected in EL and PL, but not or only at very low levels in EH and PH cells. In contrast, several genes associated with atrial myocytes were found to be weakly or not at all expressed in the EL and PL fractions, but were highly expressed in the EH and PH fractions, among them Fgf12 and Nr2f2 (encoding COUP-TFII).

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