Abstract
Background:
Myocardial infarction is the leading cause of mortality globally, due in part to the limited ability of cardiomyocytes (CMs) to regenerate. Recently, we showed that overexpression of a combination of 4 cell cycle factors, CDK1, CDK4, cyclin B1, and cyclin D1 (collectively known as 4F), induced cell division in ~15% of post-mitotic mouse, rat, and human CMs. The aim of the current study is to identify novel small molecules that further augment CM cycle induction caused by the 4F.
Methods and Results:
We performed a drug screening of 30 chemical compounds with possible cell cycle regulatory activities using 60-day-old mature hiPS-CMs overexpressing the 4F and assessed the increase in EDU (DNA synthesis marker) and PHH3 (G2-M phase marker). The top hit was N-(4,6-Dimethylpyridin-2-yl)-4-(pyridin-4-yl)piperazine-1-carbothioamide (NDPPC). NDPPC showed a dose-dependent increase in the percentage of EDU and PHH3 positive nuclei in hiPS-CMs transduced with 4F. CMs transduced with 4F and treated with 1 μM NDPPC showed a significant increase in the percentage of EDU and PHH3 compared with vehicle (V)-treated CMs (PHH3; V: 20.41±2.02%, NDPPC: 33.75±3.9%, EDU; V: 18.24±1.7%, NDPPC: 30.81±2.1% n=6, p<0.001). In Silico Drug-Target prediction showed that NDPPC could interact with p38 mitogen-activated protein kinase (P38), a critical negative regulator of mammalian cell cycle. Therefore, we overexpressed P38, in the presence and absence of NDPPC and 4F. As expected, overexpression of P38 in CMs inhibited 4F cell cycle induction, and treatment with NDPPC reversed the cell cycle inhibitory effect (in presence of 4F PHH3%; V: 20.38±0.97%, P38 overexpression+V: 11.02±1.3%, P38 overexpression+NDPPC: 23.62±2.79, n=4, P<0.001). Knockdown of P38 showed a significant increase in the percentage of EDU and PHH3 positive nuclei in 4F treated CMs, and treatment with NDPPC did not show any further increase in cell cycle induction. These data indicate that NDPPC enhances the 4F cell cycle response in CMs through inhibition of P38.
Conclusions:
NDPPC is a novel inhibitor for P38. NDPPC is a promising drug to promote CM cell cycle response to the 4F. Further work in vivo is needed to test whether NDPPC could improve the 4F effect on cardiac function after ischemic injury.