Cell culture methods

3.2.1.1 Preparation of cardiomyocytes from neonatal rats

Neonatal rats were sacrificed on postnatal day 0-3 (P0-P3) in accordance with ethical standards. Hearts were extracted using forceps and ventricular tissues were collected in cell culture dishes with ice-cooled Calcium- and bicarbonate- free Hanks with HEPES (CBFHH).

Ventricular tissues were cut in half and transferred to a new culture dish with 20 ml of CBFHH and washed one additional time to remove excess blood. The tissues were minced into small pieces (1-2 mm³) and then transferred via a 10ml pipette tip to a gentle MACS C-tube (up to ~20 neonatal rat hearts).

The following enzymes were prepared from the neonatal heart dissociation kit (for 20 neonatal rat hearts) according to the manufacturer’s instructions. Reagent preparation summarized in Table 13:

The enzyme mix 1 was preheated for 5 minutes (min) at 37°C and mixed with enzyme mix 2.

2.5 ml of the enzyme mix was transferred to the C-tube containing the tissues and incubated in an inward position with the cap down for 15 min at 37°C. The C-tube was then attached to the sleeve of a gentle MACS Dissociator and followed by the program named

‘h_Tumour_01.' Incubation steps were repeated twice to complete the digestion of tissue.

After the completions of the program, C-tubes were removed from the gentleMACS Dissociator and samples were carefully resuspended in 7.5 ml of a non-cardiomyocyte medium (NCM).

To separate the tissue from the suspension, a sieve was moistened (pore size 250 µm) with 3-4 ml of NCM. The cell suspension was transferred through the sieve, collected in a fresh cell culture dish and added to a falcon tube. Cell suspensions were centrifuged at 300 g for 5 min (20°C), and the supernatant was carefully discarded and re-suspended in 4 ml of 1x Phosphate-buffered saline (PBS), homogenized and then filtered.

The percoll gradient method was used to extract and purify only the cardiomyocytes fraction from the heterogeneous cell suspensions (consisting of a mixture of fibroblasts, cardiomyocytes, and erythrocytes). The percoll stock solution was prepared by mixing 40.5 ml of percoll (4°C) with 4.5 ml of 10x PBS. Then, a top and bottom precoll solution (TPS, BPS) was prepared, by mixing 9 ml of percoll stock with 11 ml of 1x PBS and 13 ml percoll stock with 7 ml of 1x PBS respectively. To differentiate between both solutions, 200 µl of phenol red stock solution was added to TPS. Subsequently, 4 ml of TPS was added to two 15 ml reaction tubes, and 3 ml of BPS was slowly added to the bottom while transferring the pipette upward. Next, 2 ml of cell suspension mixed with 1x PBS was slowly added and centrifuged at 750 g for 30 min at 20°C. The percoll gradient (Figure 12) was divided into three different gradients (as per density of tissue), i.e. fibroblasts, cardiomyocytes, and erythrocytes.

Figure 12 | Percoll gradient with three distinct layers consisting of fibroblasts, cardiomyocytes, and erythrocytes.

The upper 5 ml of the solution composed of fibroblasts was removed, and 2 ml of cardiomyocyte layer was carefully transferred into a new 50 ml tube and washed twice with pure DMEM cardiomyocyte medium (CM) at 300 g for 5 min at 20°C. The supernatant was discarded, and the pellet was resuspended in 10 ml of pre-warmed DMEM CM. The overgrowth of fibroblasts within cardiomyocytes was prevented by the adding 10 mM of

Fibroblasts

Cardiomyocytes

Erythrocyte

antimetabolite 5'-bromo-2'-deoxyuridine (BrdU) in the cardiomyocytes medium, which selectively inhibited the DNA synthesis and consequently cell proliferation of fibroblasts.

Cells were counted as follows: 10 µl of cell suspension was mixed with 10 µl tryphan blue and transferred to a Neubauer counting chamber. The cell number was counted in all four corner chamber, and the average value was used to quantify the cell number as shown below:

Cell quantity x 2 (dilution) x 10⁴ = cell amount/mL

Cells were seeded into 1% collagen (diluted in PBS) coated plates. For six or twelve well plates, 1 x 10⁶ or 0.5 x 10⁶ cells/well were added, respectively. The neo-natal rat cardiomyocytes (NRCM) culture was maintained at 37°C in 5% CO₂ in a humidified incubator.

The medium was exchanged after 48 hours (h).

3.2.1.2 Immunofluorescence of NRCM cells

After the specific treatment of cells, the medium was aspirated, and the cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT). The cells were then washed twice with PBS and incubated for 5 min with 0.05% Triton solution to permeabilize the cell membranes. Cells were washed twice with 1X PBS, before being incubated with 1x Roti-Immunoblock for 1 h at RT. Next, the cells were incubated overnight in the dark at 4°C with PBS containing the primary antibody against the protein of interest. Cells were then washed twice with 1x PBS and incubated overnight at 4°C in the dark with the specific fluorophore-conjugated secondary antibodies and DAPI to stain the nuclei (see Table 4 for respective dilutions). Finally, the cells were washed twice with PBS and images were taken using inverted fluorescence microscopy (Olympus).

3.2.1.3 Live cell imaging

The experiments were performed in a 6 well plate with cells incubated with 2 ml of medium.

The cells were incubated with 10² µM, 10³ µM and 10⁴ µM of H2O2 for 90 min in the climate chamber of the inverse fluorescence microscope (Olympus), keeping the ideal enviornment at 37°C, 5% CO₂, 57.37% lamp intensity and the exposure time of 20 ms. A 40x objective and a bright-field filter were used to capture the video. The time-lapse recording was setup at one frame every 20 min for a total duration of 24 h, with the first frame taken at 0 sec. The change in the morphology of cells was further analyzed with Image J.

3.2.1.4 Gene knockdown via siRNA transfection

After seeding NRCMs (day 1), the first transfection was performed after 19-20 h (day 2). For the transfection, 2 different siRNA constructs were used: a siRNA, which does not bind to mammalian mRNA to serve as a control (siRNA ctrl) and a second siRNA construct that binds to the mRNA of CTGF (siRNA ctgf) and blocks its translation. For each well (6 well plate) transfection, RNAi duplex-LipofectamineRNAiMAX complexes were first produced as follows:

(1) 9.9 µl siRNA (22nM) was gently mixed with 900 µl Opti-MEM (OM).

(2) In parallel, 9.9 µl LipofectamineRNAiMAX was mixed with 900 µl OM.

(3) Both solutions were mixed and incubated for 20 min at RT.

For the transfection, the cells were washed twice with warm PBS. Then 600 µl of 37°C warm OM was added together with 400 µl of transfection solution in each well for 6 h at 37°C. Later the cells were washed twice with warm PBS and then incubated with fresh CM (day 2). The second transfection was performed 24 h after the first transfection as described above (day 3). The next medium change was conducted after 24 h (day 4). The next day (24 h later) the cells were used for particular experiments (Figure 13).

Figure 13 | Timeline for siRNA transfection.