Mice were sacrificed on d2/d3/d7 after birth; the hearts were prepared and analyzed for H2B-mCh expression by using a macroscope (AxioZoom.V16, Zeiss). Atria and ventricles of the transgenic hearts were separated and dissociated using the Neonatal heart dissociation kit (Miltenyi Biotec) without using the gentle MACS Dissociator. The heart tissue was dissected by slowly pipetting every 15 min. 7500 cells were seeded per well on a 0.001 % fibronectin-coated 384-well µ-clear microtiter plate (Greiner). Cells were incubated in differentiation medium (see above) at 37 °C and 5 % CO2. For analysis of the binucleation index, cells were fixated after overnight adhesion to the dish.
In order to transfect postnatal CMs, ventricles of αMHC-H2B-mCh/CAG-eGFP-anillin double transgenic hearts were dissociated as described above. 5 µl of 500 nM Stock solutions of hsa-miR-199a-3p or miR mimic, Negative control #1 (Ambion, Life Technologies) were incubated with 0.2 µl Lipofectamine RNAi Max (Invitrogen) in 14.8 µl OPTI-MEM on 0.001 % fibronectin-coated 384-well µ-clear microtiter plates (Greiner) for 20 min at RT. Afterward, 7500 dissociated cells were added in a volume of 60 µl differentiation medium. Medium was changed after 48 h and the cells were further incubated for 24 h.
In order to transfect postnatal CMs, ventricles of αMHC-H2B-mCh/CAG-eGFP-anillin double transgenic hearts were dissociated as described above. 5 µl of 500 nM Stock solutions of hsa-miR-199a-3p or miR mimic, Negative control #1 (Ambion, Life Technologies) were incubated with 0.2 µl Lipofectamine RNAi Max (Invitrogen) in 14.8 µl OPTI-MEM on 0.001 % fibronectin-coated 384-well µ-clear microtiter plates (Greiner) for 20 min at RT. Afterward, 7500 dissociated cells were added in a volume of 60 µl differentiation medium. Medium was changed after 48 h and the cells were further incubated for 24 h.