Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

This protocol provides a method for isolating rod-shaped cardiomyocytes from any cardiac tissue, which can then be used to measure ploidy and nucleation status of these cardiomyocytes without manual analysis. Compared to other techniques, our protocol provides more consistent cell yields and cell morphology, and as opposed to flow cytometry, we are able to visually verify nucleation and ploidy status of cardiomyocytes. The visual demonstration of this method is important because the heart tissue division and association is easier to appreciate.

Begin by placing the euthanized mouse in a supine position, and taping down the extended limbs. Use blunt-headed scissors to cut through the chest to expose the heart. Then cut the descending aorta and inferial vena cava.

Use a peristaltic pump attached to an infusion set with a 23 gauge butterfly needle to perfuse the heart by injecting three milliliters of potassium chloride PBS solution through the left ventricle. Make sure not to pierce through the septum. Then inject 10 milliliters of 4%PFA solution for 10 minutes at a rate of one milliliter per minute.

Use scissors to remove the whole heart. And if desired, isolate a specific region. Place the tissue in a 1.5 milliliter centrifuge tube containing one milliliter of 4%PFA solution.

Incubate it on a rocker for one hour. Place the heart in a petri dish with PBS solution. Squeeze it to get rid of any PFA remaining in the ventricles and wash it in PBS.

Put the fixed heart into a new 1.5 milliliter tube containing collagenate solution. Then incubate the heart on a rocker at 37 degrees Celsius overnight. On the next day, put the heart in a 35 millimeter Petri dish containing collagenate solution, and dissociate it into one millimeter pieces with forceps or scissors.

Then use a transfer pipette to further triturate the dissociated tissue for two minutes. If tissue particles still remain, use a pipette with a narrower tip and continue trituration until the majority of the tissue is broken down. Place a 200 to 600 micrometer nylon mesh over a 15 millimeter centrifuge tube.

Add five milliliters of PBS to the dissociated cells and filter them through the nylon mesh. Wash the nylon mesh by passing an additional four milliliters of PBS. Then centrifuge the filtered solution at 10 to 100 times G for one minute.

Discard the supernatant and resuspend the pellet in 10 milliliters of PBS prior to staining. After downloading the Fiji distribution of image J, open Fiji and click on help, update, and manage update sites. Check the biomed group and IFPB plugins.

To download the dependencies plugins, ellipse split and morpho lib J.Then click close and apply changes. Next, download RStudio and open it. Copy the code from the text manuscript into RConsole's command line and press enter.

then type Y in response to all prompts to install all our dependencies. To perform image quantification, open Fiji and drag analyzenucleation. py into the status bar.

When the script editing window opens, click run in the lower left corner. A dialog box will pop up asking for the location of the output data directory. Select the folder where all analysis data, figures, and other data used by the software will be stored.

In the analysis parameters, select the location of the images to be analyzed and enter the image file name format using regular expressions. After selecting the desired settings, click OK.As images from different stages of the analysis pipeline appear on screen, inspect them to make sure that the thresholding and segmentation are occurring properly. Open analyzemultinucleatedserver.

r in Rstudio. Next, to the variable named folder name, type the file path to the output data folder selected in the last step without the final slash. Click run app in the upper left corner of the script editing window.

Use the sliders to set thresholds for the minimum valid nuclear mean intensity threshold, the minimum valid nuclear area threshold, and the maximum valid minimum ferrets diameter for cardiomyocytes. Scroll down and click apply selected thresholds then click plot intensity distribution, which will render a plot of the nucleus intensity distribution of the entire sample as well as separate subplots for each grouping variable. To account for intrasample variation, scroll down and check normalize separately by group then click calculate ploidy and plot estimated ploidy distribution.

Once the normalized whole sample graph appears, the two peak patterns should be visible. Use the sliders and select thresholds to isolate the diploid and tetraploid peaks from each other and outliers. Then scroll down and click calculate ploidy and nucleation.

Finally, click the button plot and save into results folder. This method can be used to isolate uniformly singularized cardiomyocytes that are relatively pure without contaminating cells. It is easy to implement and provides consistent cardiomyocyte yields and quality from different isolations.

Cardiomyocytes isolated with this protocol can be stained using antibodies and fluorochrome conjugated azides. To show the characteristic Z line staining pattern of sarcomeres, the cells were stained with antibodies recognizing alpha actinin. In a separate experiment, EdU was administered to mice before isolating fixed cardiomyocytes.

After isolation, the mononucleated, binucleated, and trinucleated cardiomyocytes that had undergone S-phase were detected. To identify individual nuclei, the original DNA stained image was thresholded based on intensity. Ellipses were fit to the nuclear masks, segmenting individual nuclei.

Next, the pixels of the image were partitioned into territories based upon the ellipse to which they are most proximal, and the borders of these territories were used to draw lines through nuclear clusters. A similar segmentation process was used to detect cardiomyocytes. This segmentation strategy showed that the majority of cardiomyocytes from newborn mice are mononucleated.

The frequency of mononucleated cardiomyocytes is much lower in two week old mice, where they make up about 25%of the total cardiomyocyte population. When the ploidy status of individual nuclei within cardiomyocytes was measured, a higher frequency of tetraploid nuclei was detected in juvenile mice. When isolating cardiomyocytes, it is important to verify all cell clumps have broken up off their trituration.