Protocol to identify small molecules promoting rat and mouse cardiomyocyte proliferation based on the FUCCI and MADM reporters

Summary Discovery of small molecules promoting cardiomyocyte proliferation is important for heart regeneration and related heart disease. Here, we describe a protocol to isolate neonatal rat and mouse cardiomyocytes, infect cardiomyocytes with Tnnt2-mAG-hGeminin (1/110) or Tnnt2-Cre adenovirus, and identify small molecules that promote cardiomyocyte proliferation by high-content microscopy. This protocol can be modified to investigate other pro-proliferation factors in cardiomyocytes and other cell types. For complete details on the use and execution of this protocol, please refer to Du et al. (2022).1

. Schematic of the FUCCI and MADM system (A) The FUCCI system consists of two parts: RFP red fluorescent protein fused with 30-120 amino-acid residues of human Cdt1 (hCdt1) protein expressed in G1 phase; and mAG green fluorescent protein fused with amino-acid residues at sites 1-110 of human Geminin protein (hGeminin) expressed in S/G2/M phase. Cells expressing FUCCI exhibited red fluorescence in the G1 phase, green fluorescence in the S/G2/M phase, and dual fluorescence in the G1/S transition phase. (B) The MADM system consists of two knock-in genes, which are chimeric genes consisting of the N-and C-terminal of two different fluorescent proteins, such as RFP and GFP. The chimeric N-RFP/C-GFP or N-GFP/C-RFP alleles produce two non-functional proteins before Cre-mediated recombination. During the S phase of mitosis, sister chromatids replicate and pair. If inter-chromosomal recombination occurs, functional GFP and/or RFP are produced. Green and red daughter cells or yellow and colorless daughter cells are generated after mitosis.  (Table 5).
Note: Pre-chill reagents on ice and keep on ice during the whole process.
8. Incubate the prepared assembly system in a thermal cycler and maintain at 50 C for 15 min. 9. Thaw a 50 mL tube of DH5a-competent cells on ice for 5 min. 10. Add 2 mL of the assembly reaction, gently flick the tube to mix 4-5 times. 11. Incubate on ice for 30 min. 12. Heat shock at 42 C for 90 s. 13. Place back on ice for 10 min. 14. Add 950 mL of room temperature (18 C-26 C) LB medium. Incubate in a shaker at 200 rpm at 37 C for 1 h.  a. Extract and identify plasmids using agarose gel electrophoresis. b. Confirm FUCCI reporter plasmid by sequencing with forward and reverse primers in Table 2.

Virus packaging and identification
Timing: 3 weeks OBiO Technology (Shanghai, China) produced the pAdeno-Tnnt2-mAG-hGeminin (1/110) adenovirus and determined the titer of adenovirus in HEK293 cells. They used the AdMax packaging system to produce adenovirus, which the Cre/loxP (or FLP/frt) recombinase was used to recombine shuttle plasmid carrying foreign genes and backbone plasmid in HEK293 cells. After cells appeared in cytopathic effect state, we collected cell supernatants and cell lysates, and purified the virus by cesium chloride density gradient centrifugation.
19. One day before transfection, seed HEK293 cells into 6-well plates and control the density of cells at 70%-80% when transfected. a. Dissolve 4 mg viral vector plasmid (backbone plasmid: shuttle plasmid = 1:1) in Opti-MEM medium with a total volume of 250 mL and mixed gently. b. Dissolve the transfection reagent in Opti-MEM medium with a total volume of 250 mL and mix gently. 22. Add the transfection reagent diluents to the plasmid diluent, mix it gently while adding, and then place it at room temperature (18 C-26 C) for 20 min. 23. Take out the cell culture plates, add the DNA transfection reagent complex prepared above to the cell culture plates and put it back into the incubator. 24. After 6 h, aspirate the culture medium, wash once with PBS, and add 2 mL of fresh complete medium. 25. Change the culture medium every three days. Viral plaques might appear in about 7-15 days, and collect the supernatants after complete lesions.
a. Add 500 mL 10% Nonidet P40 (NP40) to each plate to lyse cells. b. Collect cell lysates, centrifuge at 7,000 g for 10 min, discard cell fragments and collect supernatants. 28. Add 50 mL virus precipitation solution (20% PEG8000, 2.5 M NaCl) per 100 mL supernatants, and place on ice for 1 h to precipitate the virus. 29. Centrifuge the above mixture at 7,000 g for 20 min, discard the supernatant.
a. Suspend the pellets in 10 mL CsCl solution with a density of 1.10 g/mL (solvent is 20 mM Tris-HCl, pH 8.0). b. Add 2.0 mL of 1.40 g/mL CsCl solution to a Beckman ultracentrifuge tube. c. Add another 3.0 mL of 1.30 g/mL CsCl solution. 30. Add 5 mL of virus suspension. 31. Centrifuge at 159,800 g for 2.5 h at 4 C. 32. Collect Viral bands with a density between 1.30-1.40 g/mL into a dialysis bag (boil the bag with 10 mM EDTA-Na2 for 10 min before use).

Obtain the MADM reporter plasmid
Timing: 5 weeks The MADM system consists of two knock-in genes, which are chimeric genes consisting of the N-and C-terminal of two different fluorescent proteins, such as RFP and GFP. The chimeric N-RFP/C-GFP or N-GFP/C-RFP alleles produce two non-functional proteins before Cre-mediated recombination. During the S phase of mitosis, sister chromatids replicate and pair. If inter-chromosomal recombination occurs, functional GFP and/or RFP are produced. Green and red daughter cells or yellow and colorless daughter cells are generated after mitosis 5,6 ( Figure 1B). This reporter further evaluates whether small molecules promote cardiomyocyte cytokinesis by infecting cardiomyocyte-specific Tnnt2-cre adenovirus in MADM TG/GT mice. 35. Preparation of H202 vector is the same as step 1. 36. Amplify the DNA plasmids containing the Tnnt2 promoter and Cre cDNA sequences that are available in the laboratory ( Figure 4A) (seen in Data S1). 37. Extract and identify the Tnnt2 and Cre fragments by agarose gel electrophoresis ( Figure 4B). 38. Design PCR primers with overlapping sequences for the Tnnt2 promoter, Cre cDNA, and H202 vector (Table 2), the length of overlapping sequences is 20 bp. 39. Carry out PCR reactions using the primers (see Table 2 for primers, Tables 3 and 4 for the PCR system and program settings). 40. Extract and identify the DNA fragments by agarose gel electrophoresis. 41. Clone Tnnt2 and Cre fragments into the H202 vector using the NEBuilder HiFi DNA assembly reaction system (Table 5). 42. Plasmid transformation is the same as steps 9-18. 43. Extract and identify the plasmids by agarose gel electrophoresis, and confirm the Tnnt2-Cre plasmid by Sanger sequencing (Table 2). 44. OBiO Technology (Shanghai, China) produced the Tnnt2-Cre adenovirus. The packaging, identification, amplification and purification procedures of pAdeno-Tnnt2-cre virus is the same as steps 19-34.   Table 2 Primers for pAdeno-Tnnt2-Cre This paper See Table 2 Primers for MADM-ML-11 TG and MADM-ML-11 GT mice genotyping The Jackson Laboratory See Table 2 Recombinant

Reagent Final concentration Amount
Tryptone 10 g/L 10 g Yeast extract 5 g/L 5 g NaCl 10 g/L 10 g Store tryptone, yeast extract, NaCl and NaOH power at room temperature (18 C-26 C) for up to 2 years. Sterilize the LB medium after preparation. Add ampicillin to make the final concentration at 100 mg/mL before use. Store sterilized LB medium at 4 C for up to 3 weeks.

Reagent Final concentration Amount
Tryptone 10 g/L 10 g Yeast extract 5 g/L 5 g NaCl 10 g/L 10 g Note: Filter digestion solution with a 0.22 mm filter using 10 mL syringes and place on ice.
Note: The constant temperature magnetic stirrer heats up slowly. In order to ensure that the heart can be digested at 37 C after harvesting, it needs to be turned on at least half an hour in advance.

5.
Transfer 15 mL pre-cooled HBSS without Ca 2+ and Mg 2+ into three sterile 100 mm dishes and place them on ice. 6. Place 3-day-old (P3) SD rats and 75% alcohol in two boxes, and cadavers in the third box. 7. Sterilize P3 SD rats with 75% alcohol, mainly focusing on the chest. 8. Decapitate pups using sterile straight scissors, and open the chest along the sternum to remove the heart.
Note: A rat heart can obtain 4 3 10 6 cardiomyocytes. The number of hearts can be calculated according to the total number of cardiomyocytes required.
9. Collect hearts with curved scissors. a. Immediately transfer into the 100 mm dish containing pre-cooled HBSS without Ca 2+ and Mg 2+ . b. Transfer hearts to other two dishes containing HBSS with curved tweezers to remove the blood, atria, and other tissues (Figure 7).

Enzymatic digestion of ventricles
Timing: 1 h Note: Do not cut the heart into too small pieces while avoiding the adherence of tissues during digestion.
11. After 5 min, leave the ventricle tissues to stand for a minute, discard the supernatant, and add a fresh 5-mL digestion solution. 12. Repeat steps 10 and 11 to remove the red cells.
Note: Before the second digestion, cut the heart tissue into smaller pieces with straight scissors to fully remove the blood.
13. After the third digestion, transfer the supernatants to a 15 mL tube containing 5 mL high-glucose DMEM with 10% FBS and 1% penicillin-streptomycin. a. Pipette up and down 10 times using a 10 mL pipette to stop the digestion. b. Place the 15 mL tube on ice.
CRITICAL: Fully mix the supernatants and complete medium by pipetting up and down to terminate digestion.  (Figure 7). 17. Carefully discard the supernatant and re-suspend the remaining cell pellets in 20 mL highglucose DMEM containing 10% FBS and 1% penicillin-streptomycin. 18. Pipette up and down using a 10 mL pipette to ensure a homogeneous solution of single cells. 19. Pass the cell suspension through a 100 mm cell strainer. 20. Seed onto two sterilized 100 mm plastic dishes to allow the fibroblasts to adhere. 21. Incubate the cells for 2 h at 37 C in a 5% CO 2 humidified incubator.
Note: This step is designed to separate non-cardiomyocytes from cardiomyocytes. Non-cardiomyocytes in the heart are mainly fibroblasts, and fibroblasts adhere to the culture plates more rapidly than cardiomyocytes. Using differential attachment technique, we allow the cells to adhere for two hours and then collect non-adhered cells for enriching cardiomyocytes.

Timing: 1 h
These procedures are performed after culturing cardiac cells for 2 h.

After culture for 2 h.
a. Gently wash the dishes with 10 mL high-glucose DMEM containing 10% FBS and 1% penicillin-streptomycin. b. Transfer the supernatant to two 15 mL tubes. 23. Centrifuge at 200 g for 5 min at 25 C. 24. Re-suspend the pellets in 10 mL high-glucose DMEM containing 10% FBS and 1% penicillinstreptomycin. 25. Pass the cell suspensions through a 100 mm cell strainer. 26. Take 9 mL cell suspension and 1 mL 0.4% trypan blue (Solarbio, C0040), pipette up and down to mix them, and stain for 3 min. 27. Load 10 mL of the mixture into the well of a cell-counting chamber. 28. Determine the total cell number and viability by a Countess II Automated Cell Counter. 29. Prepare culture medium containing high glucose DMEM, 20 mM cytosine arabinoside, and 5% horse serum.
Note: Since cytosine arabinoside blocks DNA synthesis to inhibit non-cardiomyocytes proliferation, thus avoiding the dominant growth of non-cardiomyocytes during adherent culture.
30. Add a certain amount of culture medium according to cell viability and total cell number. a. Pipette up and down using a 10 mL pipette to ensure a homogeneous solution of single cells. b. Seed the cell suspensions on 96-well plates at 15,000 cells per well and incubate at 37 C and 5% CO 2 .

Timing: 30 min
This section describes the procedures for pAdeno-Tnnt2-mAG-hGeminin (1/110) adenovirus infection in neonatal rat ventricular myocytes (NRVMs). Note: Adenovirus should be stored as aliquots to avoid repeated freezing and thawing. After taking out the virus, place the adenovirus on ice immediately. Mix adenovirus thoroughly by pipetting up and down before adding it to the medium.

Timing: 2 h
This section describes the procedures of small-molecule treatment. All the following steps are performed in a sterile cell-culture hood, except for transferring compounds by ECHO 520.
35. Take 384-well chemical library plates from a -80 C refrigerator at least 2 h in advance. a. Thaw compounds at room temperature (18 C-26 C). b. Centrifuge them quickly with a centrifuge dedicated to these plates. 36. Prepare high-glucose DMEM with 1% penicillin-streptomycin and 0.5% FBS and as negative control (0.5% FBS with DMSO) or 10% FBS as positive control (10% FBS with DMSO).

After adenoviral infection for 24 h.
a. Aspirate the culture medium. b. Immediately add fresh culture medium with 2 mM indicated small molecules.

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CRITICAL: Do not discard the liquid in several 96-well plates at one time to avoid a great impact on the cell state caused by a long operating time. When transferring compounds with ECHO 520, the plates need to be inverted, so it is necessary to ensure that there is no liquid remaining in the plates.
38. Transfer compounds in 384-well plates in a volume of 20 nL (10 mM) to 96-well plates using ECHO 520 (Labcyte) (Figure 9). 39. Distribute 100 mL DMEM with 0.5% FBS and 1% penicillin-streptomycin to each well using Multidrop Combi (Thermo Scientific), thus making a final working concentration of 2 mM for each compound. 40. Add both negative control (0.5% FBS with DMSO) and positive control (10% FBS with DMSO) in the 96-well plates as shown in Figure 9. 41. Immediately place the 96-well plate into the incubator after adding compounds and negative and positive controls. 42. Repeat steps 37-41. 43. Seal the 384-well library plates with sealing film, flat with a roller, and return to the -80 C refrigerator.

Timing: 2-4 h
This section describes the procedures for imaging and analysis of FUCCI-positive NRVMs after smallmolecule treatment for 24 h.
44. After small-molecule treatment for 24 h, aspirate the culture medium, and stain the nuclei with Hoechst 33342.  Table 6 for parameter settings of MD ImageXpress Micro XL Imaging System). Troubleshooting 5.
Note: Acquiring at least 9 images per well and analyzing 3,000 cells per well.
Optional: Images of FUCCI [mAG-hGeminin (1/110)]-positive green fluorescent NRVMs can also be acquired using Opera Phenix (Perkin Elmer) (refer to Table 7 for parameter settings of PE Opera Phenix) and the data can be analyzed using the harmony software in Opera Phenix.
46. Count the number of FUCCI-positive NRVMs and the total number of NRVMs based on cell segmentation using MetaXpress software. 47. Calculate the percentage of FUCCI-positive NRVMs in each well. 48. Determine positive hits of small molecules based on at least 2-fold greater effects on the induction of FUCCI-positive NRVMs than the negative control ( Figure 10). Troubleshooting 6. 49. Confirm candidate small molecules (2 mM) twice to assess their effects on induction of FUCCIpositive NRVMs. Troubleshooting 7.

Determine optimal concentration of candidate compounds
Timing: 1-2 weeks  The concentration of candidate compounds in the three repeated initial screenings are 2 mmol/L, the following steps describe procedures for determining the optimal working concentration for each candidate.
Note: Vortex small molecules before and after dilution into high-glucose DMEM containing 0.5% FBS. Gently shake the 24-well plates after adding small molecules to cardiomyocytes.
54. Measure the percentage of FUCCI-positive NRVMs in each well based on cell segmentation using MetaXpress software. Troubleshooting 8 and 9.

Harvesting neonatal mouse ventricles
Timing: 12-16 h This section describes the procedures of isolating cardiomyocytes from neonatal mouse ventricles with overnight pre-digestion by trypsin.
Note: All the following steps are performed in a sterile cell-culture hood, all surgical instruments should be sterile, and the prepared reagents should be kept on ice.
55. Prepare the following surgical instruments, consumables, and reagents. a. Surgical instruments. i. Three sterile curved forceps. ii. One straight sterile scissor. iii. One sterile curved scissor. b. Consumables.
i. One 0.22 mm filter.
ii. Pre-cooled HBSS without Ca 2+ and Mg 2+ . 56. Add 10 mL filtered 0.25% trypsin with EDTA and a rotor to a sterilized bottle. Place the bottle on ice. 57. Transfer 15 mL pre-cooled HBSS without Ca 2+ and Mg 2+ into three sterile 100 mm dishes and place them on ice. 58. Place P3 MADM-ML-11 TG/GT neonatal mice and 75% alcohol in two boxes, and cadavers in the third box. 59. Sterilize P3 MADM-ML-11 TG/GT mice with 75% alcohol, mainly focusing on the chest. a. Following euthanasia, open the chest of pups along the sternum. b. Use the curved scissors to collect the ventricles.
Note: A mouse heart can isolate 2 3 10 6 cardiomyocytes. The number of hearts can be calculated according to the total number of cardiomyocytes required.
60. Transfer each heart immediately into the 100 mm dish containing pre-cooled HBSS without Ca 2+ and Mg 2+ . 61. Using the curved tweezers, transfer hearts to the second and then third dish to remove the blood and other tissues. 62. Using straight scissors to make an incision in the heart, which does not need to be completely cut apart. 63. Gently transfer hearts to the bottle containing 0.25% trypsin with EDTA. 64. Place the bottle in a 4 C refrigerator and leave overnight (12-16 h).

Timing: 4 h
This section describes the procedures of isolating mouse cardiomyocytes. Ventricles are digested by collagenase II 3-4 times. To separate the cardiomyocytes from non-cardiomyocytes, the cells are plated into 2 dishes and incubated for 2 h. After that, purified cardiomyocytes are seeded into culture plates. 7 65. After pre-digestion, prepare the following surgical instruments, consumables.

EXPECTED OUTCOMES
This protocol provides a method to discover novel small molecules promoting cardiomyocytes proliferation. An effective compound increases the percentage of mAG-hGeminin + CMs ( Figure 10) and single-color MADM CMs (Figure 11). The positive ratio calculated automatically by the software allows us to compare the ability of compounds on promoting cardiomyocyte proliferation as described. 1

QUANTIFICATION AND STATISTICAL ANALYSIS
FUCCI analysis by MetaXpress 1. After imaging by MD ImageXpress Micro XL Imaging System, analyze the images in the custom module using MetaXpress software ( Figure 12). Import the images and create the image names in different channels. 2. Use the count nuclei objects module in the software, set up the cell width and fluorescence intensity of Hoechst 33342 above the background to recognize nuclei. 3. Use the cell scoring objects module in the software, set up the threshold to the fluorescence intensity of mAG above the background to identify positive nuclei. 4. Export the total numbers of nuclei and positive nuclei. 5. Use Excel or other software to calculate the positive ratio.
Optional: FUCCI analysis by harmony. 6. Alternatively analyze the images using Harmony Software (Figure 13). Use the image analysis module in Harmony to create a new analysis and then import the images. Identify nuclei by Hoechst 33342 staining. Identify single nuclei by adjusting parameters such as nuclear area, split factor, and fluorescence contrast.
Note: To correct the vignetting effect of the images, the flatfield correction mode can be adjusted to basic or advanced.  Note: If positive cells cannot be accurately identified by the mean intensity alone, more thresholds can be set up by calculating contrast and maximum fluorescence intensity.
9. Select the population. Apply several thresholds to find the population that has a high fluorescence intensity.
Note: Since the fluorescence intensity is related to the expression level of mAG and exposure time, it is necessary to reset thresholds in different experiments to select positive cells.

MADM analysis
The analysis process for MADM cardiomyocytes is similar to the above method. However, when analyzing the number of single fluorescent cells, it is necessary to set a threshold to remove cells with fluorescence in another channel.

LIMITATIONS
The limitation of this high-throughput chemical screening system is that the screening results may be affected by several factors, such as the quality of primary cardiomyocytes, the concentration of small molecules in the primary screening, and the treatment time with small molecules. Therefore, in order to ensure consistency and reliability of the screening results, it is necessary to ensure that cultured cardiomyocytes are in a good state, consider two additional repeating screens, and optimize the small molecule concentrations and treatment times. In addition, neonatal cardiomyocytes have some degree of proliferation that is different from adult cardiomyocytes, thus the outcome of this chemical screening needs to be further verified in adult cardiomyocytes. Furthermore, this method can be used for any cell types; however, current protocol is using primary neonatal rat or mouse cardiomyocytes for the screening and characterization of the compounds. In general, any cell-specific promoters can be used for driving FUCCI and MADM reporter expression, and thus, these reporters can be used for performing chemical screening on any types of cells. Due to the depth of field (DOF) of the objectives we used in MD and PE high-content microscopy will not be able to distinguish different layers of cells like the fibroblasts and cardiomyocytes, it is necessary to exclude the possibility of compounds acting on other cells contaminated including fibroblast cells by immunostaining. We infected FUCCI and MADM viruses with cardiomyocytes or fibroblasts, and found that these fluorescent reporters were specifically expressed in cardiomyocytes but not in fibroblasts. Therefore, it's necessary to confirm the specificity of reporter gene expression when applying the FUCCI and MADM systems to other cells.

Problem 1
Tissues stick together during heart digestion, resulting in fewer viable cardiomyocytes (step 15).

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Potential solution This may be due to DNA released from lysed cells when they die, making the tissues stick together. Do not slice the heart into too small pieces and reduce the stir speed during digestion. Besides, an appropriate amount of DNase can be added to decrease tissue clumping.

Problem 2
Some cardiomyocytes do not adhere and are suspended in the culture medium (step 31).

Potential solution
Optimize digestion conditions. Pretreat the plates with L-poly-lysine or laminin. Do not move plates within 48 h of cell seeding.

Potential solution
Pipette the dissolved adenovirus up and down to mix it well. Try a new aliquot of adenovirus. This may be caused by less efficient adenoviral infection, and you may increase the virus titer or increase infection time up to 48 h.

Potential solution
This may be caused by poor quality of cardiomyocytes. Avoid over-digestion and possible mechanical damage to cardiomyocytes during digestion and plating. Excessive cell density may also cause aggregation during culture. Decrease the FUCCI virus titer.

Problem 5
Few adhered live cardiomyocytes in the middle of the well in 96-well plates (step 45).

Potential solution
This may be caused by the strong force of ultrasound or the toxicity of certain compounds to cultured cardiomyocytes during drug deployment. Dead cardiomyocytes in the middle field of the well should not be imaged and analyzed.

Problem 6
The percentage of FUCCI-positive cardiomyocytes in the positive control or negative control groups is too low (step 48).

Potential solution
This could be caused by poor quality of cultured cardiomyocytes, and so repeat the experiments by re-isolating neonatal rat or mouse cardiomyocytes. Try a new aliquot of FBS or increase the percentage of FBS in the medium. Increase the FUCCI virus titer.

Problem 7
Drug screening results are not reproducible (step 49).

Potential solution
This may be caused by poor quality of cultured cardiomyocytes, and so repeat the experiments by re-isolating neonatal rat or mouse cardiomyocytes. Check the storage conditions and lifetime of the chemical library, and repeat screening with new aliquots of compounds or order new compounds.

Problem 8
The percentage of FUCCI-positive cardiomyocytes is higher in the edge wells of 96-well plates (step 54).

Potential solution
This may be caused by the edge effect. Exclude data from edge wells during analysis, and small molecules arranged at the edge wells of 96-well plates should be rearranged to the middle wells of the plate for repeated screening.

Problem 9
Poor repeatability between replicates in the same 96-well plate (steps 54 and 96).

Potential solution
Mix cardiomyocytes well before plating by pipetting up and down to ensure that the cell density is similar between replicates. Fully mix the compounds before adding them to cardiomyocytes.