Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

All animal experiments were performed conform the National Institutes of Health guidelines and approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC).

1. Preparation of the solutions and surgical equipment

1. Prior to isolation, sterilize the surgical equipment by using 70% ethanol solution.
2. Add 2.24 g of KCl to 500 mL phosphate buffered saline (PBS) solution to obtain a final concentration of 60 mM. Store KCl-PBS solution at room temperature. Use 3 mL of KCl-PBS solution per mouse.
3. Dilute 32% paraformaldehyde (PFA) solution with PBS into to obtain final concentration of 4% PFA. Prepare 10 mL of 4% PFA in PBS per mouse. Diluted PFA solution can be stored at 4 °C for 2-3 weeks in a glass container.
   NOTE: Prepared 4% PFA solution can be stored at -20 °C for longer periods of time.
4. Prepare 1 mL of collagenase solution per mouse by adding 60 mg of collagenase, type 2 per 1 mL of PBS.

2. Perfusion and fixation of the heart

1. Anesthetize the animal by using 2-5% isoflurane with an oxygen flow rate of 1 L/min. Confirm the anesthesia by confirming lack of movement and lower rate of breathing.
   NOTE: Injecting heparin (100-500 U/kg) before euthanasia can increase the cell quality and yield by preventing blood clots, thereby allowing for more efficient perfusion of the heart with fixative.
2. Euthanize the animal according to approved methodologies.
   NOTE: We followed the American Veterinary Medical Association guidelines for the euthanasia of animals, and obtained local IACUC approval for euthanasia.
3. Place the euthanized animal in supine position, and tape down extended limbs.
4. Cut through the chest to expose the heart using blunt-end scissors. Cut descending aorta and inferior caval vein.
5. Perfuse the heart by injecting 3 mL of KCl-PBS solution through the left ventricle with a flow rate of 3 mL/min using a peristaltic pump attached to an infusion set with a 23 G butterfly needle (26 G for neonates). Make sure not to pierce through the septum.
   NOTE: Alternatively, use a needle attached to a syringe to inject solutions.
6. Perfuse the heart by injecting 10 mL of 4% PFA solution for 10 min using a peristaltic pump at a rate of 1 mL/min.
7. Remove the whole heart using scissors. After removing the heart, it is possible to isolate a specific region of the heart by incising. Place the heart, or a segment of it in a 1.5 mL microcentrifuge tube containing 1 mL of 4% PFA solution. Incubate the heart on rocker at room temperature with rocking speed between 20-30 rpm for 1 h.

3. Isolation of fixed cardiomyocytes

1. Place the heart in a Petri dish containing PBS solution. Squeeze the heart to get rid of any PFA remaining in ventricles, and wash in PBS.
2. Put the fixed heart into a new 1.5 mL microcentrifuge tube containing collagenase solution (60 mg/mL). Place the tube on rocker (20-30 rpm) at 37 °C for overnight incubation.
   NOTE: Extend the incubation time up to 1 week and replenish the collagenase solution every two days to reduce the possible variation in yield if hearts are anticipated to be fibrotic, which might require longer time of collagenase digestion to digest extracellular collagen.
3. Put collagenase solution and the heart into a 35 mm Petri dish. Dissociate the heart into 1 mm pieces by using forceps or scissors.
4. Use a transfer pipette to further triturate the dissociated tissue for 2 min. If tissue particles still remain in the dish, use a transfer pipette with narrower opening and continue trituration. Continue until the majority of the tissue is broken down.
   NOTE: Over trituration causes individual cardiomyocytes to break. Make sure not to over triturate by checking regularly under a microscope.
5. Place a 200-600 µm nylon mesh over opening of 15 mL centrifuge tube.
   NOTE: For hypertrophied cardiomyocytes, it is recommended to use 400 µm nylon mesh instead of 200 µm.
6. Add 5 mL of PBS to the Petri dish containing dissociated cells and filter the solution through nylon mesh, including tissue particles. Wash the nylon mesh by passing additional 4 mL PBS.
7. Centrifuge the filtered solution at 10-100 x g for 1 min.
   NOTE: 100 x g centrifugation will not yield 100% pure cardiomyocyte population, and some non-cardiomyocyte cells are likely to be included.
8. Discard the supernatant unless one wants to stain/evaluate non-cardiomyocytes cardiac cells as well. Resuspend the pellet in 10 mL PBS prior to staining.

4. Staining cardiomyocytes

1. Collect the cells by centrifugation at 100 x g for 1 min and add 5 mL of permeabilization solution (e.g., 0.5% Triton X-100 in PBS). Incubate for 20 min at room temperature on rocker.
   NOTE: For steps 4.1, 4.2 and 4.4 use 15 mL centrifuge tubes as it is easier to remove the supernatant without disturbing the cell pellet compared to 1.5 mL microcentrifuge tubes.
2. Collect the cells by centrifugation at 100 x g for 1 min, add 5 mL of blocking buffer (e.g., 3% bovine serum albumin [BSA] in PBS) and incubate for 30 min at room temperature on a rocker.
3. Collect the cells by centrifugation at 100 x g for 1 min and add 1 mL of primary antibody solution (in PBS) with the appropriate dilution ratio. Transfer the solution into 1.5 mL microcentrifuge tube and incubate cardiomyocytes in primary antibody solution under optimized conditions (e.g., 4 °C overnight).
4. Transfer cardiomyocytes with primary antibody solution to a 15 mL centrifuge tube and add 9 mL of PBS. Incubate the cardiomyocytes for 10 min at room temperature on a rocker.
5. Collect the cells by centrifugation at 100 x g for 1 min and add 10 mL of PBS. Incubate the cardiomyocytes for 10 min at room temperature on a rocker. Repeat this step once more.
6. Collect the cells by centrifugation at 100 x g for 1 min and add the secondary antibody solution containing DAPI. Incubate for 30 min at room temperature on a rocker, followed by repeating step 4.5 twice to wash cardiomyocytes.
7. Place the cells either on coverslips or microscope-compatible plates and proceed with imaging.
   NOTE: Images included in the manuscript were taken with 10x and 40x objectives. Lasers used were: 405 nm for DAPI, 561 nm for Alpha actinin and 640 nm for Edu.

5. Setup imaging software

NOTE: Follow along with these steps using Supplementary File 1-SoftwareScreenshots.pdf.

1. Download the Fiji distribution of ImageJ.
2. Open Fiji. Click on Help > Update… > Manage Update Sites. Check the "IJPB-plugins" and "Biomedgroup" update sites to download the dependencies plugins Ellipse Split and Morpholibj.
3. Click Close. Fiji should begin downloading the dependencies. Restart Fiji when finished.
4. Download Rstudio and open it.
5. Copy install.packages(c("ggplot2", "autothresholdr", "dplyr", "purrr", "jsonlite", "shiny")) into R console's command line and press the Enter key. Type "y" in response to all prompts to install all R dependencies (Screenshot 1 in Supplementary File 1).

6. Image quantification

1. Open Fiji and drag "AnalyzeNucleation.py" (supplied as a supplementary code file) into Fiji's status bar. This will open a script-editing window. Click Run in the lower left corner to begin it (Screenshot 2 in Supplementary File 1).
2. A dialog box will pop up (Supplementary File 1: Screenshot 3), asking for the location of the output data directory. All analysis data, figures, and other data used by this software will be stored in this folder. Another, larger dialog box will pop up, displaying all image analysis settings (Supplementary File 1: Screenshot 4).
   1. Select location of directory containing images to be analyzed.
   2. Enter the image filename format using regular expressions. Enter the image filename format, indicating which parts of the filename correspond to row, column, channel, and (optionally) site within braces, using regular expressions. Do not put spaces within the braces. Surround variable parts of the filename format in braces {}. The way files are saved depends on the imaging software, and this step will retrieve relevant information from the image filename.
      NOTE: For example, the format string
      r" Plate 1-(?P<row>[A-Za-z]+)(?P<column>[0-9]+)-(?P<channel>[A-Za-z]+).tif"
      describes a filename that starts with "Plate 1-", which is followed by one or more alphabetical letters indicating the row, which is followed by one or more digits indicating the column, which is followed by "-", which is followed by one or more letters indicating the channel, which is followed by ".tif". The letters inside the angle brackets like "<>" are variable names and are automatically copied into the data when it is collected. One of the variable names must be "<channel>"
   3. Indicate the name of channels in which the nuclear stain is visible and where the cardiomyocytes are visible. These names must be exactly as they are in the part matched by the "<channel>" variable in the regular expression filenames.
   4. Indicate how the images should be grouped using comma-separated variable names. All of the images within a given group will be opened and analyzed in one batch. For example, if the images are divided into sets for each well, and there is a well for each unique combination of row and column, then write "row, column" in this field.
      NOTE: These grouping variables must be a subset of the variables used in the format string. Do not use "channel" as a grouping variable, this will separate corresponding channel images from each other.
   5. Indicate whether or not images are stitched together in one well image or are separate for each site. In the former case, site should not be indicated in the filename format string.
   6. Choose which thresholding method to use to separate nuclei from the background. All of Fiji's standard thresholding methods are available. Test different thresholding methods to determine which works best for the image set. In this example, choose the Otsu method.
   7. Indicate whether or not the threshold should be recalculated for each site image or if the same threshold should be used for every image in the group. Indicate whether the cardiomyocyte images are brightfield or use a fluorescent marker.
   8. Indicate the cardiomyocyte thresholding method. If brightfield was chosen in the previous step, this thresholding method will be applied to edge-filtered brightfield images. Indicate whether or not the threshold should be recalculated for each site image or if the same threshold should be used for every image in the group.
   9. Indicate the number of rows of site images that cover each well. Indicate the number of columns of site images that cover each well. Indicate the minimum area of nuclei in pixels. Use a generously low minimum size, a higher and more precise threshold will be calculated in the analysis step. Indicate the minimum area of cardiomyocytes.
   10. After choosing the desired settings click OK.
3. Images resembling those found in Figure 3 and Figure 4 will appear on the screen, showing the different stages of the analysis pipeline. Inspect these images to ensure that thresholding and segmentation are occurring properly.
4. The selected results folder should now be filled with analysis data (Supplementary File 1: Screenshot 5). Files other than analysis data can safely be saved in this folder as long as their names do not begin with "cm_", "nuclei_", or "nucleilink_".

7. Data analysis

NOTE: The csv files that are produced can be analyzed manually. Each analyzed image subset produces a triplet of csv files named "nuclei(metadata).csv", "nucleilink(metadata).csv", and "cardiomyocytes(metadata),csv", where (metadata) is replaced with a sequence of name-value pairs of the form "_(name)=(value)", where (name) and (value) are sequences of alphanumeric characters derived from strings matched in the regular expression given earlier. (For example, if row and column were indicated in the filenames then strings like "_row=F" and "_column=8" will be present). The unnamed leftmost column of each nuclei and nucleilink file is a nucleus ID number. The "Min" column of the nucleilink file is the id of the cardiomyocyte that contained said nucleus wholly or 0 otherwise. The "Max" column of the nuclei is the ID of the highest-numbered cardiomyocyte that contained said nucleus in part, or 0 otherwise. The "Mean" column of the cardiomyocytes file is the cardiomyocyte id number.

1. Open "AnalyzeMultinucleatedServer.R" in Rstudio (provided as supplementary code file).
2. At the top of this file is a variable named "folderName". Next to it is a filepath. In here, type the path to the output data folder selected in the last step, without the final slash (Supplementary File 1: Screenshot 6).
3. In the upper left corner of the script editing window there should be a green arrow labeled Run App. Click this arrow. It may take some time for the data to load and for the app to pop up.
4. Initially, three gating graphs will be visible, one to indicate the minimum valid nuclear area threshold, one to indicate the minimum valid nuclear mean intensity threshold, and one to indicate the maximum valid minimum feret's diameter for cardiomyocytes. Use the sliders to set these thresholds (Supplementary File 1: Screenshot 7).
   NOTE: In each of these graphs, a large, broad peak corresponding to valid nuclei or cardiomyocytes should be present, flanked by broad tails representing debris or erroneous segmented cardiomyocytes. Use the thresholds to cut one tail of each of the peaks off.
5. Scroll down. Click the button Apply Selected Thresholds (bottom of Supplementary File 1: Screenshot 7).
6. Click the button Plot Intensity Distribution. This will render plot of the nuclear intensity distribution of both the entire sample and separate subplots for each grouping variable.
   NOTE: For example, if <row> and <column> grouping variables were entered into the regular expression in the Fiji dialog, plots indicating the intensity distribution by row and by column will appear here (Supplementary File 1: Screenshot 8). If illumination and staining conditions were constant across the different parts of the sample, these plots should all clearly show two intensity peaks, a dimmer, taller one for the diploid nuclei and a brighter, shorter one for the tetraploid nuclei.
7. Intrasample variation will result in this pattern not being visible in the whole-sample plot and there being great variety in the location of the diploid and tetraploid peaks by row, column, or other grouping variable. In the latter case, scroll down check the checkbox Normalize Separately by group to account for this variation (Supplementary File 1: Screenshot 9).
8. Click the button Calculate Ploidy (Supplementary File 1: Screenshot 9). Click the button Plot Estimated Ploidy Distribution. Graphs will appear in the empty windows to the right. In the normalized whole-sample graph, the two-peak pattern should be visible if it wasn't before.
9. Select thresholds to isolate the diploid and tetraploid peaks from both each other and outliers using the sliders (Supplementary File 1: Screenshot 9). Scroll down. Click the button Calculate Ploidy and Nucleation (Supplementary File 1: Screenshot 10).
10. Click the button Plot and Save Into Results Folder. The plot saved into the selected results folder will also appear in this interactive window (Supplementary File 1: Screenshot 10).