The overall goal of this protocol is to quantify cardiomyocyte turnover using long term nucleoside labeling and flow cytometry to quantify the neo cardiomyocyte nuclei while accounting for polyploidization of the nuclei. This method can be used to address key questions in the field of cardiomyocyte regeneration and it can be used as a screening tool to quantify changes in the dynamics of cardiomyocyte generation. The main advantage of this protocol is it allows a sensitive and accurate quantification of neo cardiomyocyte generation, irrespective of the cellular source and controlling for polyploidation.
24 hours after the sixth EdU injection, place the treated animal in the supine position and use surgical scissors to make a skin incision from the mid abdomen to the diaphragm. Next holding the sternum away from the body cavity bilaterally cut the diaphragm to expose the heart. Then lift the heart slightly and cut the major blood vessels of the outflow tract to dissect the heart from the thoracic cavity.
Immediately upon harvest, place the hearts into individual 15 mL centrifuge tubes containing 10 mL of PBS chilled to four degrees Celsius. Then transfer the samples to individual 10 cm petri dishes and use a scalpel to cut each heart into two pieces in a sagittal orientation. Next gently squeeze the heart pieces with a pair of forceps.
Remove both pieces of heart and place them into a petri dish containing 20 mL of fresh PBS. Repeat once again with a new petri dish and 20 mL of fresh PBS. Place both parts of the hearts into the same 1.5 mL microcentrifuge tube and use a 25 gauge needle to make a small hole in the lid of each tube to prevent the lid from opening when the heart has processed.
Then label each tube and freeze the cardiac tissues in a container of liquid nitrogen. For each heart to be analyzed, incubate 36 mL of one percent BSA and PBS in an ultra centrifuge tube. Discard the solution after 30 minutes.
When the tubes have air dried, add 10 mL of sucrose gradient solution to each tube. After individually mincing the liquid nitrogen frozen heart samples with the scalpel, transfer the tissue fragments from each heart into individual 50 mL conical tubes containing 15 mL of cell lysis buffer. Homogenize the samples for 15 seconds at 25, 000 rpm with a probe homogenizer at room temperature.
Then add 15 more mL of lysis buffer to each tube and transfer the samples one at a time to a 40 mL dounce with a large clearance pestle. Perform 10 strokes with the pestle and filter the cell slurries through a 100 micron strainer followed by a 40 micron cell strainer into individual 50 mL centrifuge tubes. Then collect the nuclear cardiac pellets by centrifugation and resuspend the cells in 25 mL of sucrose gradient solution each.
Layer the cells on top of the nuclei free sucrose gradient in the prepared ultra centrifuge tubes and centrifuge the layers using a swing out tube rotor. Following the centrifugation, resuspend the nuclei pellets in 1, 300 microliters of nuclei storage buffer and transfer the nuclei to a 1.5 mL microcentrifuge tube labeled PCM1. Transfer 300 microliters of each suspension from the PCM1 tubes into a new microcentrifuge tube and add 700 microliters of fresh nuclei storage buffer and label ISO control.
Next label the PCM1 nuclei with anti-PCM1 antibody and the ISO control nuclei with rapid IGG isotype control antibody overnight at four degrees Celsius. The next day spin down the samples and resuspend the pellets in one mL of fresh nuclei storage buffer for a second centrifugation under the same conditions. After the second spin resuspend the pellets in one mL of fresh nuclei storage buffer and incubate the nuclei with one microliter of a fitzy conjugated F(ab'2 fragment from a goat anti rabid IGG antibody for one hour at four degrees Celsius in the dark.
To evaluate the degree of EdU incorporation centrifuge the PCM1 and ISO control labeled nuclei suspensions and resuspend the pellets in one mL of 1%BSA PBS two times for two consecutive washes. After the second wash, replace the supernatant with 100 microliters of EdU fixative and incubate the samples for 15 minutes at room temperature in the dark. At the end of the fixation wash the nuclei two more times in one mL of PBS BSA then incubate the samples with 100 microliters of saponin based permeablization solution at room temperature.
After 15 minutes, add 500 microliters of freshly prepared EdU labeling cocktail to each tube for a 30 minute incubation at room temperature. At the end of the incubation, spin down the samples three times washing the nuclei in one mL of PBS BSA during the second two centrifugations. Finally label the samples with 400 microliters of DNA staining solution.
Immunohistological analysis of the pulsed hearts demonstrates the presence of PCM1 expressing cardiomyocytes that have incorporated EdU. However, the data obtained using immunohistological methods can be misinterpreted as other cell types that have incorporated EdU can be misidentified as cardiomyocytes. In a study of mdx and control mice, the flow cytometry analysis detailed in this protocol enables a rapid quantification of neo cardiomyocyte nuclear generation while also excluding the cells that have incorporated EdU to depolyploidization.
Similar to previously published data, in this representative experiment an increase in the rates of neo cardiomyocyte nuclei generation in the mdx mouse hearts compared to age matched controls was observed. Once mastered this technique can be completed in four hours over the space of two days. When performing this procedure it's important to remember both the EdU and PCM1 negative controls so the gates can be set accurately during the analysis.
This technique is also compatible with a number of additional downstream immunohistological techniques allowing nucleocyte incorporation to be quantified in a number of different cardiac cell types. This technique has been used in the field of cardiovascular research to study changes in cardiomyocyte regeneration in the mdx model of non ischemic cardiomyopathy. After watching this video you should have a good understanding of how to rapidly quantify neo cardiomyocyte nuclei, watch the count in for polyploidation to screen for changes in cardiomyocyte regeneration and turnover.
