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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 embryonic atrial and ventricular cardiomyocytes.(A) E15.5 CMs were purified by sorting of whole-heart suspensions into PI-/ERBB-2+ and PI-/ERBB-2- cells. (B) E15.5 mouse hearts were dissociated and sorted into ERBB-2+/ITGA6low (EL) and ERBB-2+/ITGA6high (EH) cells. (C) Density plots, distribution of the cells before and after sorting. Dot plots, co-staining of the fractions with pure antibodies against α-actinin and MLC-2a (labeled with APC rat anti-mouse IgG1 or IgG2ab). (D) Sorted fractions were plated on fibronectin-coated dishes. Cells were attached after one day; most of them flattened but some still round. Immunofluorescence analysis of plated cells one day after sorting in terms of α-actinin cross-striation (mid panel) and of Ki-67 expression (right panel). (E) Statistical analysis of the CM subtype isolation with regard to the content of α-actinin and MLC-2a before and after sorting. Data are expressed as mean ± SD, n = 4. t-test for paired samples with** p ≤ 0.01, *** p ≤ 0.001 vs. before sorting.
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pone.0143538.g004: Selective enrichment of embryonic atrial and ventricular cardiomyocytes.(A) E15.5 CMs were purified by sorting of whole-heart suspensions into PI-/ERBB-2+ and PI-/ERBB-2- cells. (B) E15.5 mouse hearts were dissociated and sorted into ERBB-2+/ITGA6low (EL) and ERBB-2+/ITGA6high (EH) cells. (C) Density plots, distribution of the cells before and after sorting. Dot plots, co-staining of the fractions with pure antibodies against α-actinin and MLC-2a (labeled with APC rat anti-mouse IgG1 or IgG2ab). (D) Sorted fractions were plated on fibronectin-coated dishes. Cells were attached after one day; most of them flattened but some still round. Immunofluorescence analysis of plated cells one day after sorting in terms of α-actinin cross-striation (mid panel) and of Ki-67 expression (right panel). (E) Statistical analysis of the CM subtype isolation with regard to the content of α-actinin and MLC-2a before and after sorting. Data are expressed as mean ± SD, n = 4. t-test for paired samples with** p ≤ 0.01, *** p ≤ 0.001 vs. before sorting.

Mentions: We analyzed the expression of known CM surface markers such as ALCAM and VCAM-1 [6,9] and included ERBB-2 into the analysis due to its important role in CMs and heart development [22]. In full agreement with earlier reports, single cell analysis of mouse hearts of various developmental stages confirmed highly regulated expression of ALCAM and VCAM-1 with restriction to CMs at E11.5. Moreover, we found CM-restricted expression of ERBB-2 at E15.5 (S3 Fig, S1 Methods). In fact, ERBB-2 flow sorting of E15.5 hearts resulted in a potent enrichment of CMs in the ERBB-2+ fraction (94.6% ERBB-2+/α-actinin+); only 1.7% CM were retained in the ERBB-2- fraction (Fig 4A). We therefore combined ERBB-2 with ITGA6 and sorted embryonic hearts into ERBB-2+/ITGA6low (EL) and ERBB-2+/ITGA6high cells (EH) (Fig 4B). As depicted in Fig 4C, this led to a high enrichment of CMs in both populations: the α-actinin frequency increased from 75% in the unsorted whole-heart preparation to 99% and 98% in EL and EH, respectively. As indicated by MLC-2a labeling, EL and EH highly differed in their composition. From initial 9.4% in the whole heart MLC-2a frequency was elevated up to 70% in the EH fraction and reduced down to 1.6% in the EL population.


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 embryonic atrial and ventricular cardiomyocytes.(A) E15.5 CMs were purified by sorting of whole-heart suspensions into PI-/ERBB-2+ and PI-/ERBB-2- cells. (B) E15.5 mouse hearts were dissociated and sorted into ERBB-2+/ITGA6low (EL) and ERBB-2+/ITGA6high (EH) cells. (C) Density plots, distribution of the cells before and after sorting. Dot plots, co-staining of the fractions with pure antibodies against α-actinin and MLC-2a (labeled with APC rat anti-mouse IgG1 or IgG2ab). (D) Sorted fractions were plated on fibronectin-coated dishes. Cells were attached after one day; most of them flattened but some still round. Immunofluorescence analysis of plated cells one day after sorting in terms of α-actinin cross-striation (mid panel) and of Ki-67 expression (right panel). (E) Statistical analysis of the CM subtype isolation with regard to the content of α-actinin and MLC-2a before and after sorting. Data are expressed as mean ± SD, n = 4. t-test for paired samples with** p ≤ 0.01, *** p ≤ 0.001 vs. before sorting.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143538.g004: Selective enrichment of embryonic atrial and ventricular cardiomyocytes.(A) E15.5 CMs were purified by sorting of whole-heart suspensions into PI-/ERBB-2+ and PI-/ERBB-2- cells. (B) E15.5 mouse hearts were dissociated and sorted into ERBB-2+/ITGA6low (EL) and ERBB-2+/ITGA6high (EH) cells. (C) Density plots, distribution of the cells before and after sorting. Dot plots, co-staining of the fractions with pure antibodies against α-actinin and MLC-2a (labeled with APC rat anti-mouse IgG1 or IgG2ab). (D) Sorted fractions were plated on fibronectin-coated dishes. Cells were attached after one day; most of them flattened but some still round. Immunofluorescence analysis of plated cells one day after sorting in terms of α-actinin cross-striation (mid panel) and of Ki-67 expression (right panel). (E) Statistical analysis of the CM subtype isolation with regard to the content of α-actinin and MLC-2a before and after sorting. Data are expressed as mean ± SD, n = 4. t-test for paired samples with** p ≤ 0.01, *** p ≤ 0.001 vs. before sorting.
Mentions: We analyzed the expression of known CM surface markers such as ALCAM and VCAM-1 [6,9] and included ERBB-2 into the analysis due to its important role in CMs and heart development [22]. In full agreement with earlier reports, single cell analysis of mouse hearts of various developmental stages confirmed highly regulated expression of ALCAM and VCAM-1 with restriction to CMs at E11.5. Moreover, we found CM-restricted expression of ERBB-2 at E15.5 (S3 Fig, S1 Methods). In fact, ERBB-2 flow sorting of E15.5 hearts resulted in a potent enrichment of CMs in the ERBB-2+ fraction (94.6% ERBB-2+/α-actinin+); only 1.7% CM were retained in the ERBB-2- fraction (Fig 4A). We therefore combined ERBB-2 with ITGA6 and sorted embryonic hearts into ERBB-2+/ITGA6low (EL) and ERBB-2+/ITGA6high cells (EH) (Fig 4B). As depicted in Fig 4C, this led to a high enrichment of CMs in both populations: the α-actinin frequency increased from 75% in the unsorted whole-heart preparation to 99% and 98% in EL and EH, respectively. As indicated by MLC-2a labeling, EL and EH highly differed in their composition. From initial 9.4% in the whole heart MLC-2a frequency was elevated up to 70% in the EH fraction and reduced down to 1.6% in the EL population.

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