Functional and structural phenotyping of cardiomyocytes in the 3D organization of embryoid bodies exposed to arsenic trioxide | Scientific Reports -…

Cell lines

R1 mESCs (kindly provided by Dr. Nagy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada) and mouse STO-SNL2 cells (STO, American Type Culture Collection CRL-2225) were cultivated as previously described48.

R1 mESCs were induced to differentiate into embryoid bodies (EBs) by removing the Leukemia Inhibitory Factor from the culture medium (differentiation medium), using the hanging drop method24. Briefly, for EBs formation, about seventy 20 L droplets of culture medium containing 103 mESCs were plated on the lid of p55 Petri dishes. On day 3 of culture, the developing EBs were transferred on 0.1% agarose-coated tissue dishes (Corning) and from day 5, about 58 EBs were plated in single 1.9 cm2 well or 2 EBs in single 0.3 cm2 well or onto 22mm gelatin-coated 35mm glass bottom dish (Greiner Bio-One) and cultivated for up to 15days.

ATO (Sigma, cat. n. 11,099) was dissolved in 0.1N NaOH in milliQ water (Millipore) to a final concentration of 100M. This solution was added to the culture medium on day 15 of differentiation to a final concentration of 0.1, 0.5 or 1.0M. As control samples, cells were cultured in the presence of 0.01N NaOH (CTR). ATO or NaOH were left in the culture medium for 72h, until day 18 of differentiation. Three independent experiments were performed for each of the assays described below.

About 20 EBs were plated onto 22mm gelatin-coated Glass Bottom Dish (WillCo Wells), cultured up to day 15 and then exposed for 72h to 0.1, 0.5 or 1.0M ATO. Then, dishes were transferred into the culture chamber of a Nikon BioStation IM, at 37C and 5% CO2, for video recording. For each experiment, AVI videos of the beating syncytia were recorded from 10 randomly chosen CTR or ATO-exposed samples, using the Snagit software and further analyzed with the Video Spot Tracker (VST) program used to track the motion of spots in AVI videos. Videos were then processed as previously described27,28. Briefly, 12 markers were positioned on randomly-chosen beating cardiomyocytes onto the first video frame. Then, for each marker, the spatial coordinates x and y, expressed in [pixel], and the temporal coordinate t, expressed in [s], were registered frame by frame. Using an image processing algorithm developed by one of us (L.F., see Fassina et al.29), the marker trajectories (characterized by vectors describing the movement, e.g., displacements and velocities) were mathematically calculated. Chronotropy was measured by counting the displacement peaks during the contraction movement; dynamic and kinematic inotropies were evaluated in terms of contraction force by the Hamiltonian mechanics (where force is demonstrated to be the gradient of total energy) and in terms of the maximum contraction velocities, respectively; ergotropy (consumption of energy to sustain the contraction movement) was estimated as mean kinetic energy of the beating syncytia.

Cellular ATP content was determined using the ATPLite 1 step Luminescence ATP Detection Assay (PerkinElmer), following the manufacturers instructions. Briefly, 9 EBs for CTR or 9 for each ATO-exposed samples were washed twice with 100l of PBS, lysed with 50l of lysis buffer per well in a 96-well microplate (PerkinElmer), and shaked for 2min at 700rpm with an orbital shaker. Fifty l of substrate solution was then added and incubated for 10min in the dark. Luminescence was measured with a luminometer (Perkin Elmer Victor 2). Each sample was measured in triplicate. Three independent experiments were performed.

Following 15days of differentiation, about 12 EBs, plated onto 22mm gelatin-coated Glass Bottom Dish (WillCo Wells), were exposed for 72h to 0.1, 0.5 or 1.0M ATO. On day 18, CTR and ATO-exposed EBs were washed twice with 1X PBS and fixed in 4% cold paraformaldehyde in 1X PBS for 24h. EBs were then permeabilized with 1% Triton X-100 in 1X PBS (PBT) for 30min for 3 times and then blocked for 1h with 10% FCS, 0.2% sodium azide in 1X PBS (Blocking buffer) at room temperature. EBs were washed twice in blocking buffer and incubated with mouse anti-cardiac -actinin (1:800; Sigma) or with anti-mouse cardiac isoform of Troponin T (1:200; Thermo Fisher Scientific), and anti-rabbit Connexin 43 (Cx43; 1:75; Cell Signalling) primary antibodies, diluted in 1% FCS, 0.4% Triton X-100 (PBS-MT), 0.2% sodium azide in 1X PBS, for 24h, at 4C on gentle rotation. In parallel, EBs were stained with TRITC-conjugated phalloidin (1:1000; Sigma). Then, EBs were rinsed 3 times for 45min in 1% Triton X-100, 10% FCS in 1X PBS, 3 times for 10min in 1X PBT, 3 times for 45min in 1% FCS, 0.2% sodium azide in 1X PBS and 3 times for 10min in 1X PBT. EBs were then incubated for 40h at 4C on gentle rotation device with secondary antibodies diluted in Blocking buffer [Alexa fluor 488-conjugated anti-mouse IgG 1:500 (Molecular Probes); Alexa fluor 647-conjugated anti-rabbit IgG (Molecular Probes)]. After 3 washes with PBT, nuclei were counterstained with 0.2g/ml DAPI for 2h and mounted in VECTASHIELD Mountain Medium (Vector Labs). All images were acquired with a 40X oil immersion objective with a Leica TCS SP8 confocal microscope.

Quantification of phalloidin and Cx43 immunostaining signals in non-cardiomyocyte cells was performed using Fiji software (http://imagej.nih.gov/ij/). Briefly, 50 regions of interest (ROIs) for CTR and 50 for 1.0M ATO-exposed samples of 2500 m2 for phalloidin or of 0.968 m2 for Cx43 were drawn and the fluorescent mean intensity value recorded.

On day 18, after 72h ATO exposure, a total of 300 EBs for CTR or 300 for each ATO-exposed samples were washed two times with 1X PBS and collected by centrifugation at 500rpm for 5min. CTR and ATO-exposed cells were lysed in 50mM TrisHCl pH 8, 150mM NaCl, 0.02% sodium azide, 1% Triton X-100 and 100mg/ml PMSF. The concentration of the proteins was assayed using the Bradford method with a spectrophotometer (Bio-Rad). Samples were aliquoted and stored at 80C until usage.

Ten g proteins were separated on 812% polyacrylamide gels and transferred on membranes (BioRad), overnight (40V, 4C). Membranes were blocked and incubated with primary antibodies, as reported in Table 1S. After washes, the appropriated secondary antibodies were used to reveal the primary antibodies for 30min at 37C, in agitation (Table 1S). Chemiluminescent detection was performed using the Westar C (Cyanagen), according to the manufacturers instructions. The blots were imaged with ChemiDoc XRS system (Bio-Rad) and acquired with Quantity One software (Bio-Rad). Densitometric intensities of the bands were determined with Image J software (National Institute of Health, http://imagej.nih.gov/ij/).

For the detection of different proteins on the same samples, the membranes were incubated with a stripping-buffer, containing 2% SDS, 62.5mM Tris HCl pH 6.8 and 0.8% -mercaptoethanol (Sigma), for 45min at 50C in agitation. Then, the membranes were washed in tap water for 5min in agitation and three more times using TBS-T for 10min. At the end of the stripped procedure, membranes were reused for the detection, as previously described.

On day 18, after 72h ATO exposure, RNA was extracted using the GenElute Mammalian Total RNA Kit (Sigma, according to the manufacturers instruction) from about 250 EBs for CTR or for each 0.1, 0.5, 1.0M ATO-exposed samples. All traces of DNA contamination were eliminated using the On-column DNaseI Digestion Kit (Sigma, according to the manufacturers instruction). Reverse transcription and quantitative Real-Time PCR reactions were performed as previously described14. The sequences of specific primers used are reported in Table 2S. -2-microglobulin gene expression was used for sample normalization14.

Cardiomyocytes for single-cell immunofluorescence analysis were isolated from CTR, 0.1, 0.5 or 1.0M ATO-exposed samples, as described in Neri et al.31. Briefly, a total of 160 EBs (40 for CTR and 40 for each ATO concentration) were mechanically detached from the wells, centrifuged at 500rpm for 5min and resuspended in 1ml of low Ca2+-medium (120mM NaCl, 5.4mM KCl, 5mM sodium pyruvate, 20mM glucose, 20mM taurine, 10mM HEPES) for 15min at room temperature. Then, cells were dissociated in low Ca2+-medium, supplemented with 1mg/ml collagenase and 30mM CaCl2, for 30min at 37C. EBs dissociation was completed by vortexing the suspension for 1min, at high speed. Cells were then centrifuged at 500rpm for 5min, resuspended in culture differentiation medium with 0.1, 0.5 or 1.0M ATO and seeded on 0.1% gelatin-coated coverslips in 24-well culture dishes. Following attachment, cardiomyocytes were fixed with 4% cold paraformaldehyde in 1XPBS for 20min at room temperature and maintained in 1X PBS at 4C until usage.

For immunostaining, cells were permeabilized with 0.1% Triton X-100 in 1X PBS, then incubated with anti-cardiac-actinin (1:800 in 1X PBS; Sigma) primary antibody for 1h at 37C, rinsed thrice with 1X PBS, and then in an AlexaFlour 488-conjugated anti-mouse IgG (1:500 in 1X PBS; Molecular Probes) 1h at 37C. After three washes with 1X PBS, nuclei were counterstained with 0.2g/ml DAPI and mounted in VECTASHIELD Mountain Medium (Vector Labs). Control experiments with the secondary antibody only were also carried out.

The SarcOmere Texture Analysis (SOTA) algorithm developed by Sutcliffe et al.30 was employed to quantify the texture organization of the sarcomeres and their length and width, and the area, eccentricity, circularity, and elongation of cardiomyocytes.

EBs, immunostained for cardiac -actinin as described above, were used to determine the sarcomere organization. Within their 3D structure, sixty single stack images, derived from three independent experiments (twenty for each experiment), of CTR or of each ATO-exposed sample were acquired with a 63X oil immersion objective plus 1.5 digital zoom with a Leica TCS SP8 confocal microscope. Stacks were obtained with axial distances of 0.5m.

Specific ROIs were drawn around portions of cardiomyocyte sarcomeres (Fig. 5S). Using the MATLAB software (The MathWorks, Inc.), sarcomere organization was evaluated via a Fourier score (based on Fourier transforms), a Gabor score (based on Gabor filters), and a Haralick texture feature (Haralick correlation being an indicator of organization). In detail, the Fourier transform converts an image to the frequency domain to assess the repeating structure of the sarcomere; the Gabor filter is able to detect image edges, in our study the sarcomeres edges; the Haralick correlation is calculated from the gray level co-occurrence matrix of an image30. Sarcomere length and width were also calculated following Sutcliffe et al.30.

For the calculation of Fourier Score, Gabor Score, Haralick Correlation, sarcomere length and width parameters, a total of 290 ROIs for CTR or for each of the three ATO concentrations were analyzed.

Cardiomyocytes were dissociated and immunostained for cardiac -actinin as described above. Fifty cardiomyocyte images of CTR or of each ATO-exposed sample were acquired with 100X oil immersion objective with an Olympus BX60 fluorescence microscope, captured with a DP72 camera (Olympus) and processed using cellSens 1.4.1 software.

The morphological parameter analyzed with SOTA were the cardiomyocyte area, eccentricity, circularity, and elongation as described in Sutcliffe et al.30. Specifically, the perimeter of each cardiomyocyte was delimited to determine its area along with the minor and major axis lengths. These four parameters were used to evaluate the following cell shape parameters:

Eccentricity is comprised between 0 and 1, where a value of 0 represents a circumference, a value greater than zero but less than 1 represents an ellipse.

Circularity equal to 1 represents a circumference; a circularity<1 represents an ellipse.

Elongation is a form factor; an elongation equal to 1 represents a circumference; an elongation>1 represents an ellipse.

For area, circularity, eccentricity, and elongation parameters, a total of 65 cells for CTR or for each of the three ATO concentrations were analyzed.

EBs were immunostained for -actinin, as described above. Stacks were obtained from at least 20 core samples for each experiment, for a total of 60 core samples for CTR and each of the three ATO concentrations. The acquisition of each core sample starts from the first visible nucleus on the top of a selected region of the EB and continues until the surface of the dish, to which cells are attached, thus acquiring for the entire thickness of the EB (Fig. 5S). Images were acquired with a 40X oil immersion objective with a Leica TCS SP8 confocal microscope. Stacks were obtained with axial distances of 0.5m. To determine the volume occupied by cardiomyocytes in the core samples, a stereology approach based on the extrapolation of 3D information from the 2D planar sections of a tissue was applied. The ImageJ software tool called SUM (https://imagej.nih.gov/ij/), able to sum the cardiac -actinin-related pixel intensities of all optical sections along the Z direction of a core sample, was applied. Then, the Integrated Density (IntDen) tool was applied to calculate the integral of the fluorescence intensity in an area where it is not homogeneously distributed. Then, using an area in which cardiomyocytes were absent, the mean background value was evaluated with the same approach. Finally, the CTF was determined applying the following formula:

$${text{CTF}} = left( {text{IntDen of EB core sample SUM}} right) - {text{n*}}left( {text{mean of background IntDen}} right)$$

where n is the number of images of each single core sample.

All data are presented as meansstandard deviation (SD), except for the syncytium contractile properties that are expressed as mean95% confidence interval for the differences between means. Data were analyzed by the one-way ANOVA (significance level of 0.05) followed by the post hoc LSD test.

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Functional and structural phenotyping of cardiomyocytes in the 3D organization of embryoid bodies exposed to arsenic trioxide | Scientific Reports -...