Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

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.


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. Introduction that could distinguish between different cardiomyocyte subtypes at E11.5 of mouse heart development [17].
Based on a surface-marker screen on E13.5 mouse hearts, we introduce ITGA6 to discriminate between atrial and ventricular CMs due to differential protein expression intensities. Using flow cytometry, we unambiguously show a direct correlation of ITGA6 with CM subtype-specific intracellular proteins in the same cell at this stage of mouse heart development.
In contrast to previously described markers, we show that differential ITGA6 expression persists throughout development up to the adult heart. Based on this, we developed isolation strategies using ITGA6 expression in combination with either ERBB-2 co-labeling or a preenrichment step to selectively enrich the desired CM subtype out of mixed cell populations.

Ethical statement
This study was conducted in accordance with the German animal protection law and with the European Communities Council Directive 2010/63/EU for the protection of animals used for experimental purposes. All experiments were approved by the North Rhine Westphalia State Agency for Nature, Environment and Consumer Protection. The permission is issued to Miltenyi Biotec GmbH (84-02.05. 20.12.215).

Dissociation of embryonic and neonatal heart tissue
The animals were maintained under specific pathogen-free conditions according to the recommendations of the Federation of European Laboratory Animal Science Association. Embryonic (CD1, wild-type, E11.5 -E17.5) or neonatal (P2) whole hearts or atrial and ventricular fractions were pooled and dissociated either manually using Collagenase B as described [18] or automated using the Neonatal Heart Dissociation Kit (Miltenyi Biotec) in combination with the gentleMACS™ Dissociator (Miltenyi Biotec). Blood cells were removed by red blood cell lysis or labeled with anti-mouse CD45 and anti-Ter119 antibodies to exclude them from the flow cytometry analysis.

Antibody-based surface marker screen
Mouse hearts were isolated from embryos (CD1, wild-type, E13.5). The atria were mechanically removed from the ventricles. Both tissues fractions were independently dissociated and screened for surface markers with our in-house antibody library against mouse surface markers. 10 4 atrial or 10 5 ventricular cells were incubated with FcR Blocking Reagent (Miltenyi Biotec) and then resuspended in 100 μL of staining solution (antibody plus PBS/0.5% BSA/2 mM EDTA). Incubation for 10 min at 4°C was eventually followed by an incubation of the secondary antibody. To exclude blood cells from the analysis, each sample was co-stained with fluorochrome-coupled antibodies against murine CD45 and Ter119 (all Miltenyi Biotec). A counterstaining with PE rat anti-mouse CD166 (ALCAM) (eBioscience, 1:100) was applied as far as possible. The entire list of tested antibodies, fluorochromes, staining titers, and respective co-labeling are provided in S1 Table. Partial or full overlap with ALCAM expression indicated candidate markers for CMs (enrichment) or non-myocytes (depletion). The ratio of cells labeled with the antibody of interest served as a secondary criterion. The labeled cells were resuspended in an adequate volume of staining buffer for flow cytometry analysis. Staining and measurement were performed in 96-well U bottom plates. Samples were measured on a MACSQuant 1 Analyzer (Miltenyi Biotec). Doublets, cell debris and dead cells (identified by propidium iodide, Sigma, 20 μg/mL) were excluded from the analysis. Analysis gates were set according to unstained or secondary antibody controls. Labeling frequencies were calculated by subtraction of the background value of the corresponding control sample. Negative values were set to 0.00.

Gene expression analysis of sorted embryonic and neonatal cardiomyocytes
E15.5 ERBB-2 + /ITGA6 low and ERBB-2 + /ITGA6 high cells as well as P2 ITGA6 low and ITGA6 high CMs were sorted from mouse hearts in four independent experiments each and subjected to gene expression analysis using Agilent Whole Mouse Genome Oligo Microarrays (8x60K, Design ID 028005) as previously described [19,20]. In brief, total RNA was isolated using the NucleoSpin RNA Kit (Macherey-Nagel) and amplified and labeled with the Low Input Quick Amp Labeling Kit (one-color, Agilent Technologies). Cy3-labeled fragmented cRNA was hybridized over night (17 hours, 65°C) to the microarrays. The obtained data were quantile normalized and transformed to logarithms to the base of 2. Hierarchical clustering was conducted using the MultiExperimentViewer 4.9.0. Differentially expressed genes were determined by a combination of statistical tests (ANOVA, Benjamini-Hochberg correction for multiple testing p 0.05, Tukey post-hoc test p 0.05) and effect size (3x). The mRNA data discussed in this publication has been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE57131 (http://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE57131).
Whole-cell patch clamp analysis of sorted embryonic cardiomyocyte subtypes E15.5 hearts were flow sorted using a BD Influx or BD Aria III, re-plated and cultivated for two days (IMDM w/ 20% FCS, 0.1 mM non essential amino acids, 50 μg/ml penicillin and streptomycin, 0.1 mM β-mercaptoethanol). Action potentials from spontaneously beating CMs were recorded using the patch clamp technique in the current clamp configuration, as reported previously [21]. All experiments were performed at 37°C. Cells were tested for voltage activated channels by running 250 ms lasting depolarizing ramps from -120 mV to 50 mV (holding potential: -50 mV) in the voltage-clamp mode. Statistical analysis was performed using unpaired t-tests with Graph Pad Prism 5 and LabChart 7 (AD Instruments) software for data analysis. Cells were analyzed from three independent flow sorting experiments and the results are expressed as mean ± standard error of the mean (SEM); a value of p 0.05 was considered significant.

Antibody-based surface marker screen on embryonic mouse heart
We considered embryonic mouse hearts of E13.5 as a good model system to study developing CM subtypes because the four-chambered heart is established at this developmental stage while the CMs are still immature. In order to identify CM subtype-specific surface markers, we mechanically removed atria from ventricles. Both tissue fractions were independently dissociated and analyzed (S1A and S1B Fig). Using this model system we carried out a surface marker screen with 170 anti-mouse antibodies on both tissue fractions. The entire list of tested antibodies, staining conditions and frequencies are provided in S1 Table. From the candidates we first selected antibodies that labeled more than 10% of non-blood cells in at least one of the cardiac chamber fractions resulting in a list of 51 antibodies ( Fig 1A). Next we chose antibodies either detecting co-expression with ALCAM, previously described as CM marker [6,7,8], or marking a strong difference of antigen expression between atria and Selective Identification and Isolation of Cardiomyocyte Subtypes ventricles. Validation experiments analyzed potential co-expression of cardiac troponin T and the respective surface marker candidates.
As expected, we observed a full overlap of ALCAM and VCAM-1, previously described as transient surface marker of embryonic CMs [9]. We could show that at this stage of development 80% of the CMs in both fractions co-express VCAM-1 ( Fig 1B). Apart from VCAM-1 we noticed additional candidates associated with endothelium. The liver sinusoidal endothelial cell marker LSEC (CD146) was found on E13.5 CMs in both fractions, but its expression was neither restricted to CMs nor differentially expressed in the atrial and ventricular fraction (S1C Fig). The endothelial cell marker Endoglin (CD105) although expressed in embryonic heart was absent from CMs at this stage (S1C Fig). Therefore, both markers were not further investigated.
We also noticed expression of several integrin chains among the 51 markers. Whereas ITGA2 (CD49b) was not expressed on embryonic CMs (S1C Fig), two integrin α chains, α5 (ITGA5, CD49e) and α6 (ITGA6, CD49f), were strongly expressed on and highly differed between the atrial and ventricular fraction. Validation experiments revealed a bright ITGA6 staining on CMs in the atrial cell fraction ( Fig 1C, left panel). In contrast to this, co-labeling with the antibody against ITGA5 resulted in a distinct staining of the ventricular CMs ( Fig 1C, right panel). Therefore, we focused on ITGA5 and ITGA6 to further investigate their differential cardiac expression pattern.

ITGA5 and ITGA6 are differentially expressed on atrial and ventricular cardiomyocytes
After the initial screening and validation experiments on chamber fractions, we next analyzed expression of ITGA5 and ITGA6 in whole heart preparations. As exemplary shown in Fig 2 virtually all E13.5 CMs expressed ITGA6 (Fig 2A) and ITGA5 (Fig 2B). ITGA6-expressing CMs segregated into a subpopulation with low fluorescence intensity (ITGA6 low ) and one with high fluorescence intensity (ITGA6 high ) (Fig 2A): 77.9% of CMs (24.4% of heart cells) were ITGA6 low and 22.1% of CMs (6.9% of heart cells) were ITGA6 high . Similar to ITGA6, ITGA5positive CMs could be subdivided into two populations based on their fluorescence intensity. As depicted in Fig 2B, 64.6% of CMs (21.7% of heart cells) were ITGA5 high and 35.4% of the CMs (11.9% of heart cells) were ITGA5 low .
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.
Next we co-labeled ITGA5 or ITGA6 with chamber-specific MLC-2a and MLC-2v antibodies ( Fig 3B). Gating boundaries of MLC-2a and -2v staining were set according to the secondary antibody control (S2A Fig). MLC-2a + cells co-expressed ITGA6 high (6.2%) and ITGA5 low (6.5%), whereas MLC-2v expression correlated with ITGA5 high (21.4%) and ITGA6 low (20.7%). This corresponded to our previous finding, and we concluded that antibodies against both surface markers could be used to separate atrial and ventricular embryonic CMs based on fluorescence intensity after respective antibody labeling. We focused on ITGA6 and investigated its expression at various stages of cardiogenesis ( Fig 3C, S2B Fig). Antibody labeling revealed a differential ITGA6 fluorescence intensity of atrial and ventricular CMs as early as E11.5, which increased with age up to P2.
In addition, we tested atrial and ventricular fractions from P8 as well as from adult mouse hearts in order to evaluate whether differences in fluorescence intensity between atrial and ventricular CMs were restricted to the developing or conserved in adult heart. As shown in S2C   As flow cytometry analysis indicated that ITGA6 was not restricted to CMs, but could be found on other heart cells, we sought to either combine the subtype-specific marker with a surface marker labeling all CMs or to apply a depletion strategy to remove all cardiac nonmyocytes.

Selective enrichment of embryonic and neonatal cardiomyocyte subtypes
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-2fraction ( Fig 4A). We therefore combined ERBB-2 with ITGA6 and sorted embryonic hearts into ERBB-2 + /ITGA6 low (EL) and ERBB-2 + /ITGA6 high cells (EH) ( Fig  4B). As depicted in Fig 4C, this led to a high enrichment of CMs in both populations: the αactinin 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.
The sorted cells were viable, could be cultivated and exhibited spontaneous contractions 24 h after plating (Fig 4D, S1 and S2 Videos). CM identity was confirmed by the typical cross-striated α-actinin staining pattern in all cells of both fractions (Fig 4D). Cell cycle activity of the sorted cells was analyzed by Ki-67 staining: 28 out of 205 nuclei expressed Ki-67 in the EL fraction (= 13.7%) and 42 out of 238 in the EH fraction (= 17.7%). Walsh and co-workers [23] determined a Ki-67 labeling index of~12% for E14.5 mouse hearts which confirms our findings.
As ALCAM, VCAM-1 as well as ERBB-2 expression decreased after birth (S3 Fig), the twomarker 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. ITGA6 low (PL) and ITGA6 high 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).

Embryonic and neonatal ITGA6 high and ITGA6 low -sorted cardiomyocytes display atrial and ventricular gene expression patterns, respectively
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 ITGA6 low (EL, PL) and ITGA6 high 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, Selective Identification and Isolation of Cardiomyocyte Subtypes but were highly expressed in the EH and PH fractions, among them Fgf12 and Nr2f2 (encoding COUP-TFII).
Differentially expressed genes were determined by a combination of statistical tests and effect size (3-fold). 329 genes were higher expressed in EH and PH compared to EL and PL, 129 genes were lower expressed (S2 and S3 Tables). A list of selected genes is provided in heatmap format (Fig 6B) and in Table 1. As expected, atrial genes (e.g. Sln, Gja5, Nppa, Tbx5) were overrepresented in EH and PH, whereas ventricle-specific genes (e.g. Lbh, Myh7) were underrepresented. Corresponding to the flow cytometry data, we found an enrichment of the gene encoding MLC-2a, Myl7, as well as of Itga6 in EH and PH. No difference was observed for genes encoding general CM surface markers such as Alcam, Vcam1 and Erbb2 as well as for Itga5. Altogether, expression analysis confirmed selective enrichment of atrial and ventricular genes in the sorted fractions. Embryonic ITGA6 high and ITGA6 low -sorted cardiomyocytes display atrial and ventricular-like action potentials, respectively 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 + /ITGA6 low (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 + /ITGA6 high (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].
We also analyzed the functional expression of voltage dependent currents by applying voltage ramps. These experiments illustrated that all major components of voltage activated currents, namely Na + , Ca 2+ -and inwardly-and outwardly rectifying currents are expressed in EL Selective Identification and Isolation of Cardiomyocyte Subtypes and EH CMs (Fig 7D). These data demonstrate the physiological viability of the sorted cells as well as the successful separation into atrial-and ventricular-like CMs [9,28].

Discussion
To date, several methods have been described to isolate CMs, e.g. by the surface markers VCAM-1 [9,10] or ALCAM [6,7]. The first surface marker-dependent isolation of a CM subpopulation, namely pacemakers, has been described based on transient ALCAM expression [8]. The isolation of atrial and ventricular CM populations at E11.5 using integrin expression has just recently been reported [17]. Herein we present a novel surface marker-dependent purification of atrial and ventricular CMs from embryonic as well as neonatal mouse hearts.
Our antibody-based surface marker screen on embryonic mouse hearts validated previously published CM surface markers. In addition, we identified ERBB-2 as a specific surface marker for CMs at day E15.5. It is well known that ERBB-2 plays an important role in early and later Table 1. List of fold-change values of selected genes with general or subtype-specific expression in mouse cardiomyocytes.

Gene
Name Ref.

E15.5 P2
genes that are higher expressed in atrial CM

Hey2
Hairy-related transcription factor 2; HRT-2 [11,14] -30.8 -67.1 Positive fold-change values indicate a higher abundance in ITGA6 high as compared to ITGA6 low -sorted cells, negative values demonstrate a higher abundance in ITGA6 low -sorted cells in comparison to ITGA6 high . Differential gene expression was assumed for fold-change values 3.0 or -3.0.
doi:10.1371/journal.pone.0143538.t001 stages of cardiogenesis [22,29] and we show here that ERBB-2-based flow sorting even facilitates the purification of embryonic CMs with purities of > 94%. Main α chain integrins in CMs are α1, α5, and α7, which heterodimerize with integrin β1 to form collagen, fibronectin and laminin-binding receptors, respectively. Additionally described is the expression of α6, α9 and α10 [16]. Nevertheless, integrins are found throughout the body, and subunit expression varies e.g. with developmental stages and cell-type. While ITGA5 is found mostly on embryonic and neonatal CMs, it is replaced by integrin α7 in the adult heart. Data from in situ hybridization and immunohistochemistry indicated expression of ITGA6 in the developing heart [30].
In our antibody screen, we discovered that atrial and ventricular CMs are characterized by differential expression of ITGA6 and ITGA5 at E13.5. This is in line with the findings of Tarnaswki and co-worker who used a triplet to integrin α chains for the isolation of CM subtypes a E11.5 [17]. Using qRT-PCR and electrophysiology they classified the flow sorted populations as atrial (ITGA6 + ITGA1 -ITGA5 -), ventricular compact (ITGA6 -ITGA1 + ITGA5 + ), and ventricular trabecular cells (ITGA6 + ITGA1 + ITGA5 + ).
Our single cell analysis of mechanically separated E13.5 hearts revealed that the expression level of a single surface marker, i.e. either ITGA6 or ITGA5, can discriminate between atrial and ventricular CMs. Additionally, we could directly relate the variation in integrin expression levels to the expression of MLC-2a (ITGA6 high , ITGA5 low ) and MLC-2v (ITGA6 low , ITGA5 high ) More importantly, we show that the differential ITGA6 expression in atrial and ventricular CMs is not a transient feature as it is for ITGA5 but persists throughout heart development and could even be demonstrated in adult hearts. This makes ITGA6 stand out from all previously identified CM surface markers.
Although flow cytometry-based discrimination of CM subtypes requires only one surface marker, ITGA6, CM purification needs either a second surface marker or a CM pre-enrichment step. We unequivocally demonstrate that ERBB-2 is a suitable second marker for discrimination of CMs and non-CMs at E15.5 and that magnetic pre-depletion of non-myocytes facilitates the isolation of highly pure atrial and ventricular CMs from neonatal hearts. Based on the expression kinetics we suggest VCAM-1 or ALCAM as additional CM markers for the isolation of CM subtypes of earlier embryonic stages. In fact, an efficient purification of CMs from E9.5 -E12.5 mouse hearts by flow sorting of VCAM-1 + /PECAM-1cells has been described [9]. Combined with ITGA6 we assume this could be used as an alternative purification strategy to isolate CM subtypes at these stages as well.
Apart from flow cytometry, we could also show the segregation of atrial and ventricular CMs by microarray analysis. In particular, the genes encoding key transcription factors of atrial and ventricular identity [11] such as the orphan nuclear receptor COUPT-TFII and HEY2 were found among the most differentially regulated genes and were absent from the ITGA6 low and ITGA6 high sorted fractions, respectively. Therefore, we were wondering whether ITGA6 is linked to the transcriptional networks that render atrial and ventricular identity. Recently, it has been described that COUP-TFII determines atrial identity since it promotes atrial (Tbx5) gene expression by tethering to SP1 binding sites and it represses ventricular gene expression (Hey2, Irx4) [11,29]. Therefore, we analyzed the Itga6 core promoter in terms of regulatory elements using the TRANSFAC database (Rel. 3.3 06-01-1998). Based on an 85.0 threshold, 18 high-scoring transcription factor binding sites were identified in the Itga6 promoter, among them seven Sp1 sites (S4 Table). This could provide a first possible link of ITGA6 to the transcription factor network specifying atrial identity. Further investigations like reporter gene and ChiP assays are required to better understand the potentially functional role of ITGA6 during establishment of CM subtypes.
In summary, we established a novel method to selectively identify and isolate primary atrial and ventricular CMs from mouse heart based on distinct expression levels of ITGA6. Our newly developed method facilitates the enrichment of viable CM subtypes suitable for any downstream application like physiological or biochemical analysis and in vitro cultivation. Although beyond the scope of our study, differential expression levels of ITGA6 may as well be detected on PSC-derived CMs or subtypes and as well enable their identification and selective enrichment. Identity of atrial and ventricular cells was analyzed by qRT-PCR with regard to transcript levels of the ventricle-specific Myl2, encoding MLC-2v, the atrium-specific Myl7 (MLC-2a) and Nppa (ANF) as well as the endothelium-specific Cdh5 (VE-cadherin). Gene expression was calculated relative to Gapdh and analyzed using the ΔΔCT method (S1 Methods). The fold change is calculated as a relative change of atrial versus ventricular expression. Bar graph shows mean ± SD, n = 5. (C) Density plots, co-labeling of surface markers LSEC, Endoglin, and ITGA2 with intracellular cardiac troponin T. Density plots, whole-heart cell suspensions were co-labeled with antibodies against ITGA6 and α-actinin. Histograms, ITGA6 expression gated on α-actinin + cells of whole-heart (top row) and of mechanically separated atrial (mid row) and ventricular cells (bottom row). At all investigated stages atrial and ventricular CMs increasingly differ in ITGA6 expression intensity. (C) Histograms, cardiomyocytes were purified from P8 wholeheart, atria, and ventricles, and labeled with an antibody against ITGA6. There was only one peak detectable in the whole-heart preparation (left plot) but staining of isolated fractions revealed a remaining difference in ITGA6 expression intensity between the two fractions (right plot). Overlay: red peak = ventricular fraction, blue peak = atrial fraction. (D) Histogram, cardiomyocytes were isolated from adult atria and ventricles (S1 Methods), and were co-labeled with antibodies against ITGA6 and cardiac troponin T. There is a difference in ITGA6 expression intensity. Overlay: red peak = ventricular fraction, blue peak = atrial fraction. Abbreviations: A-area, H-height, FSC-forward scatter, SSC-side scatter, APC-allophycocyanin, PE-Rphycoerythrin, FITC-fluorescein. (TIF) S3 Fig. Temporal expression pattern of ALCAM, VCAM-1 and ERBB-2 in the developing mouse heart. Representative flow analysis of mouse hearts of different developmental stages (E11.5 -P2). Density plots, whole-heart cell suspensions were co-labeled with antibodies against α-actinin (X-axis) and the surface markers ALCAM, VCAM-1 or ERBB-2 (Y-axis). Gates were set to corresponding unstained controls. As illustrated, cardiac expression of the markers was highly regulated during development. Cardiomyocyte-specific expression of ALCAM and VCAM-1 at E11.5 as well as of ERBB-2 at E15.5 is indicated by the red rectangle. (TIF) S4 Fig. Correlation matrix showing the relationship of gene expression profiles. In four independent experiments embryonic and neonatal mouse heart cells were flow sorted and collected for gene expression analysis. The matrix was generated by unsupervised hierarchical clustering of pair-wise correlation coefficients (Pearson). Correlation coefficients are indicated by their color from 0.94 (black) to 1.0 (yellow). As depicted in the heat-map, hierarchical clustering of the complete dataset by experiments resulted in a clear separation of the different sample groups. EL = E15.5 ERBB-2 + /ITGA6 low , EH = E15.5 ERBB-2 + /ITGA6 high , PL = purified P2 CM ITGA6 low , PH = purified P2 CM ITGA6 high ; n = 4. (TIF) S1 Methods. Supplemental methods and materials. (DOC) S1 Table. List of tested antibodies, staining conditions and frequencies of the Antibodybased surface marker screening. (XLS) S2 Table. List of differentially expressed genes with a higher abundance in embryonic and neonatal ITGA6 high sorted cells (alphabetical order). Positive fold change values indicate a higher abundance in ITGA6 high as compared to ITGA6 low -sorted cells; differential gene expression was defined as a fold change value 3.0. (XLSX) S3 Table. List of differentially expressed genes with a higher abundance in embryonic and neonatal ITGA6 low sorted cells (alphabetical order). Negative fold change values indicate a higher abundance in ITGA6 low as compared to ITGA6 high -sorted cells; differential gene expression was defined as a fold change value -3.0. (XLSX) S4 Table. List of transcription factor binding sites in the Itga6 core promoter. (XLSX) S1 Video. Contracting flow sorted ERBB-2 + /ITGA6 low cells. E15.5 mouse hearts were flow sorted into ERBB-2 + /ITGA6 low cells (EL) and plated on fibronectin-coated dishes. 10 s videos were recorded from spontaneously beating cells 24 h after plating. (AVI) S2 Video. Contracting flow sorted ERBB-2 + /ITGA6 high cells. E15.5 mouse hearts were flow sorted into ERBB-2 + /ITGA6 high cells (EH) and plated on fibronectin-coated dishes. 10 s videos were recorded from spontaneously beating cells 24 h after plating. (AVI)