Neonatal mice (1-day postnatal, P1) were collected and placed in a separate cage. P1 mice were anaesthetised at a low temperature (hypothermia can provide anaesthesia by reducing nerve conduction and synaptic transmission). Subsequently, the mice were placed under a stereomicroscope, and the skin was disinfected with a cotton swab dipped in betadine. Microsurgical scissors cut the skin from left to right between the third and fourth ribs of mice, and the rib cage was exposed after the blunt separation of the intercostal muscles. Surgical scissors removed about 15% of the apex tissue from the left ventricle. After surgery, the heart was gently returned to the chest using a saline swab, and the ribs and skin were closed layer by layer using surgical sutures (6-0). Finally, the mice were placed under an infrared physiotherapy lamp to restore body temperature. When the skin was rosy and the limbs began to move, the mice were put back in the cage. After the operation of all mice, all mice were sent to the mother mice. The sham group performed the same procedure described above without AR. A specific scheme of combined adenovirus injection in AR of mice (AR + Ad5-cTNT-INK4ai or AR + Ad5-cTNT-NC): 10% Trypan blue solution with PBS, and adenovirus stock solution was diluted 1 : 9 in Trypan blue solution (the total amount of injected virus was 1∗109 pfu/virus). A microsyringe with a 36 G needle was used to drain the virus diluent. The needle was injected slowly at three locations around the apex of the heart (anterior, lateral, and posterior wall: 2 μl each). Under the microscope, the edge of the myocardium can be seen to be stained blue, indicating successful injection.
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