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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

Functional subtype characterization of sorted cells confirms selective enrichment of atrial and ventricular cardiomyocytes.(A) Top graph, typical ventricular-like action potential (AP) of a cell from the EL group. Bottom graph, typical atrial-like AP from a CMs of the EH group. (B) Distribution of the cells in the two sorted groups. (C) Statistical analysis of AP parameters: left, action potential duration at 90% of repolarization (ADP90); mid, maximum rate of rise of the AP (max dV/dt); right, maximum diastolic polarization (MDP). Data are expressed as mean ± SEM. *** p ≤ 0.001 EL vs. EH. (D) Representative voltage ramps recordings from an E15.5 ERBB-2+/ITGA6low CM (left) and an E15.5 ERBB-2+/ITGA6high CM (right) show functional expression of inward and outward current components. 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.g007: Functional subtype characterization of sorted cells confirms selective enrichment of atrial and ventricular cardiomyocytes.(A) Top graph, typical ventricular-like action potential (AP) of a cell from the EL group. Bottom graph, typical atrial-like AP from a CMs of the EH group. (B) Distribution of the cells in the two sorted groups. (C) Statistical analysis of AP parameters: left, action potential duration at 90% of repolarization (ADP90); mid, maximum rate of rise of the AP (max dV/dt); right, maximum diastolic polarization (MDP). Data are expressed as mean ± SEM. *** p ≤ 0.001 EL vs. EH. (D) Representative voltage ramps recordings from an E15.5 ERBB-2+/ITGA6low CM (left) and an E15.5 ERBB-2+/ITGA6high CM (right) show functional expression of inward and outward current components. Abbreviation: EL = E15.5 ERBB-2+/ITGA6low, EH = E15.5 ERBB-2+/ITGA6high, PL = P2 ITGA6low, PH = P2 ITGA6high.

Mentions: To determine the viability and the functional subtype of the different CM fractions, action potentials (AP) were recorded from sorted E15.5 cells by patch-clamp experiments. 85.7% of the CMs (total n = 14) sorted with ERBB-2+/ITGA6low (EL) exhibited typical AP shape and long AP duration characteristic for ventricular-like cells; in this group only one cell had an atrial-like and one a pacemaker-like phenotype (Fig 7A and 7B). The CM population sorted with ERBB-2+/ITGA6high (EH) demonstrated a prevalently atrial-like phenotype with a very short AP duration (87.5%, total n = 16 cells); only two cells displayed a pacemaker-like and none a ventricular-like AP (Fig 7A and 7B). The most important AP parameters for both populations were quantified (Fig 7C) and yielded the following results: APD90: 132.6 ± 11.8 ms (EL), 47.4 ± 4.5 ms (EH); MDP: -60.1 ± 2.1 mV (EL), -61.2 ± 1.8 mV (EH); Max dV/dt: 16.3 ± 0.8 V/s (EL), 14.0 ± 0.7 V/s (EH). The two populations differed significantly for APD90. These values correspond to previous studies of murine embryonic CMs [9,27].


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)

Functional subtype characterization of sorted cells confirms selective enrichment of atrial and ventricular cardiomyocytes.(A) Top graph, typical ventricular-like action potential (AP) of a cell from the EL group. Bottom graph, typical atrial-like AP from a CMs of the EH group. (B) Distribution of the cells in the two sorted groups. (C) Statistical analysis of AP parameters: left, action potential duration at 90% of repolarization (ADP90); mid, maximum rate of rise of the AP (max dV/dt); right, maximum diastolic polarization (MDP). Data are expressed as mean ± SEM. *** p ≤ 0.001 EL vs. EH. (D) Representative voltage ramps recordings from an E15.5 ERBB-2+/ITGA6low CM (left) and an E15.5 ERBB-2+/ITGA6high CM (right) show functional expression of inward and outward current components. Abbreviation: EL = E15.5 ERBB-2+/ITGA6low, EH = E15.5 ERBB-2+/ITGA6high, PL = P2 ITGA6low, PH = P2 ITGA6high.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664422&req=5

pone.0143538.g007: Functional subtype characterization of sorted cells confirms selective enrichment of atrial and ventricular cardiomyocytes.(A) Top graph, typical ventricular-like action potential (AP) of a cell from the EL group. Bottom graph, typical atrial-like AP from a CMs of the EH group. (B) Distribution of the cells in the two sorted groups. (C) Statistical analysis of AP parameters: left, action potential duration at 90% of repolarization (ADP90); mid, maximum rate of rise of the AP (max dV/dt); right, maximum diastolic polarization (MDP). Data are expressed as mean ± SEM. *** p ≤ 0.001 EL vs. EH. (D) Representative voltage ramps recordings from an E15.5 ERBB-2+/ITGA6low CM (left) and an E15.5 ERBB-2+/ITGA6high CM (right) show functional expression of inward and outward current components. Abbreviation: EL = E15.5 ERBB-2+/ITGA6low, EH = E15.5 ERBB-2+/ITGA6high, PL = P2 ITGA6low, PH = P2 ITGA6high.
Mentions: To determine the viability and the functional subtype of the different CM fractions, action potentials (AP) were recorded from sorted E15.5 cells by patch-clamp experiments. 85.7% of the CMs (total n = 14) sorted with ERBB-2+/ITGA6low (EL) exhibited typical AP shape and long AP duration characteristic for ventricular-like cells; in this group only one cell had an atrial-like and one a pacemaker-like phenotype (Fig 7A and 7B). The CM population sorted with ERBB-2+/ITGA6high (EH) demonstrated a prevalently atrial-like phenotype with a very short AP duration (87.5%, total n = 16 cells); only two cells displayed a pacemaker-like and none a ventricular-like AP (Fig 7A and 7B). The most important AP parameters for both populations were quantified (Fig 7C) and yielded the following results: APD90: 132.6 ± 11.8 ms (EL), 47.4 ± 4.5 ms (EH); MDP: -60.1 ± 2.1 mV (EL), -61.2 ± 1.8 mV (EH); Max dV/dt: 16.3 ± 0.8 V/s (EL), 14.0 ± 0.7 V/s (EH). The two populations differed significantly for APD90. These values correspond to previous studies of murine embryonic CMs [9,27].

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