P16(INK4a) Regulates ROS-Related Autophagy and CDK4/6-Mediated Proliferation: A New Target of Myocardial Regeneration Therapy

Neonatal mice achieve complete cardiac repair through endogenous myocardial regeneration after apical resection (AR), but this capacity is rapidly lost 7 days after birth. As an upstream inhibitor of cyclin-dependent kinase 4/6- (CDK4/6-) mediated cell cycle activity, p16(INK4a) is widely involved in regulating tumor and senescence. Given that p16(INK4a) had a significant negative regulation on cell proliferation, targeting cardiomyocytes (CMs) to inhibit p16(INK4a) seems to be a promising attempt at myocardial regeneration therapy. The p16(INK4a) expression was upregulated during perimyocardial regeneration time. Knockdown of p16(INK4a) stimulated CM proliferation, while p16(INK4a) overexpression had the opposite effect. In addition, p16(INK4a) knockdown prolonged the proliferation time window of newborn myocardium. And p16(INK4a) overexpression inhibited cell cycle activity and deteriorated myocardial regeneration after AR. The quantitative proteomic analysis showed that p16(INK4a) knockdown mediated the cell cycle progression and intervened in energy metabolism homeostasis. Mechanistically, overexpression of p16(INK4a) causes abnormal accumulation of reactive oxygen species (ROS) to induce autophagy, while scavenging ROS with N-acetylcysteine can alleviate autophagy and regulate p16(INK4a), CDK4/6, and CyclinD1 in a covering manner. And the effect of inhibiting the proliferation of p16(INK4a)-activated CMs was significantly blocked by the CDK4/6 inhibitor Palbociclib. In summary, p16(INK4a) regulated CM proliferation progression through CDK4/6 and ROS-related autophagy to jointly affect myocardial regeneration repair. Our study revealed that p16(INK4a) might be a potential therapeutic target for myocardial regeneration after injury.