FOXK1 / K2 Induce Metabolic Reprograming Coupled With Cardiomyocyte Proliferation

Background The limited self-renewal ability of adult CMs is not enough to replenish for the lost CMs,which is an important cause of heart failure caused by various cardiovascular diseases.Promoting the regeneration of the pre-existing CMs in the injured heart to replenish the lost CMs,thereby saving heart function,is considered to be the most effective approach to treat patients with heart failure.The mouse heart has the ability to regenerate after being injured for a short time after birth,but this regenerative potential disappears 7 days after birth.With the loss of regenerative potential after birth,the metabolic pattern of CMs changes,which is specifically manifested as a down-regulation of glycolysis and an up-regulation of aerobic metabolism.This is considered to be an important reason for the cell cycle arrest of CMs.Regulating the metabolism of CMs has become one of the key measures to regulate CMs proliferation.Recent studies have shown that FOXK1 and FOXK2 induce cellular glycolysis and suppress oxidation of pyruvate in the mitochondria by regulating a variety of metabolic enzymes.Whether FOXK1 and FOXK2 can regulate CMs metabolism and proliferation,and then affect heart regeneration is a direction worth exploring.Objective To explore the role and mechanism of FOXK1 and FOXK2 in CM metabolism and proliferation,and heart regeneration.Methods and Results 1.FOXK1 / K2 promote glycolysis and inhibit oxidative phosphorylation of CMs The results of the glycolysis inhibitor 2-DG inhibited the glycolysis of CMs showed that the proliferation of CMs was inhibited and it caused a significant increase in the m RNA and protein expression of FOXK1 and FOXK2.After interfering with the expression of FOXK1 or FOXK2 in CMs,the Seahorse Cell Energy Metabolism Analyzer test was performed.The results showed that knockdown of either Foxk1 or Foxk2 in CMs decreased glycolysis and increased oxidative phosphorylation of CMs and overexpression of either FOXK1 or FOXK2 in CMs promoted glycolysis and suppressed oxidative phosphorylation of CMs.2.FOXK1 / K2 play an important role in CMs proliferation and heart regeneration.The results of in vitro cell experiments showed that knockdown of either Foxk1 or Foxk2 inhibited CMs proliferation,and overexpression of either FOXK1 or FOXK2 increased CMs proliferation.Subsequently,the specific overexpression of FOXK1 or FOXK2 in cardiomyocytes by injecting adeno-associated virus(AAV-9)into mice at postnatal 1 day(P1)could promote the proliferation of P14 CMs,and increase the number of CMs and the proportion of mononuclear diploid CMs.Next,the CM-specific knockout Foxk1 mice and Foxk2 systemic knockout mice were introduced respectively,and a heart injury model(MI)were constructed at P1.The results showed that after CMs specifically knocked out Foxk1 or systemic knocked out Foxk2,P28 mice could not achieve heart regeneration after MI at P1,and the heart function was significantly worse at P28 and the proliferation of CMs was significantly lower at P6 than that of the control group.Furthermore,adult mice were given AAV-9 injections after MI to make CMs specifically overexpress FOXK1 or FOXK2.The results showed that the heart function was significantly restored and the size of MI was significantly smaller at 56 days after MI compared with the control group after CMs specifically overexpressed either FOXK1 or FOXK2.The results of 14 days after MI showed that CMs specifically overexpressed either FOXK1 or FOXK2 significantly promoted CMs proliferation compared with the control group.3.Explore the mechanisms of FOXK1 / K2 regulating the metabolism and proliferation of CMs 3.1 Hif-1α regulates the metabolic reprogramming of CMs induced by FOXK1 / K2 In order to screen the downstream regulatory genes of FOXK1 or FOXK2,we used sequencing after CUT & Tag to analyze the sequence directly bound by FOXK1 or FOXK2. The results suggested that both FOXK1 and FOXK2 can bind to the Hif-1α promoter region,which was further confirmed by post-Chip q RT-PCR(Chip-q RT-PCR).The results of q RT-PCR and WB showed that FOXK1 and FOXK2 could regulate the m RNA and protein expression of Hif-1α.Furthermore,CMs overexpressed either FOXK1 or FOXK2 concomitantly using si RNA to knock down the expression of Hif-1α in vitro,and then performed metabolic analysis and detected proliferation of CMs.The results showed that knockdown of Hif-1α could attenuate the overexpression of either of FOXK1 or FOXK2 increased glycolysis and decreased oxidative phosphorylation and the increased proliferation of CMs.The above results indicate that both FOXK1 and FOXK2 can regulate the metabolism of CMs by regulating the expression of Hif-1α.3.2 DNA damage response pathway is involved in CMs proliferation regulated by FOXK1 / K2 The ROS production of CMs was detected by ROS detection kit,and we found that knockdown of either Foxk1 or Foxk2 after separation of CMs in vitro caused an increase in ROS production of CMs.In the CM-specific knockout Foxk1 mice and Foxk2 systemic knockout mice,ROS production in CMs after P1 MI was also significantly increased at P6,meanwhile,DNA oxidative damage significantly increased and DNA damage response pathways were activated.Furthermore,the results of WB showed that the expression of cell cycle inhibitor Wee1 kinase increased after the knockdown of either Foxk1 or Foxk2 of CMs in vitro.The above results indicate that the metabolism of CM regulated by FOXK1 and FOXK2 further affects the proliferation of CMs by influencing DNA damage response pathway.3.3 FOXK1 directly regulates Ccnb1 to promote CMs proliferation Further analysis of the CUT & Tag sequencing data found that FOXK1 can bind to the Ccnb1 promoter region,which was further confirmed by Chip-q RT-PCR.Next,we found that FOXK1 could regulate the expression of Ccnb1 through q RT-PCR and WB.Finally,FOXK1 was overexpressed in CMs concomitantly with Ccnb1 knocked down by si RNA in vitro,and then the proliferation of cardiomyocytes was detected.The results showed that the increased CMs proliferation caused by overexpression of FOXK1 is inhibited by knockdown of Ccnb1.The above results indicate that FOXK1 can also affect CM proliferation by directly regulating the expression of Ccnb1.3.4 FOXK2 directly regulates Cdk1 to promote CMs proliferation It was also found that FOXK2 can bind to the promoter region of Cdk1 through in-depth analysis of the CUT & Tag sequencing data,which was further confirmed by Chip-q RT-PCR.Next,through q RT-PCR and WB detection,we found that FOXK2 could regulate the expression of Cdk1.Finally,FOXK2 was overexpressed in CMs concomitantly with Cdk1 knocked down by si RNA in vitro,and then the proliferation of CMs was detected.We found that the increase of CM proliferation caused by overexpression of FOXK2 is inhibited by knockdown of Cdk1.The above results indicate that FOXK2 can also regulate the proliferation of CMs by directly regulating the expression of Cdk1.Conclusions In this study,we demonstrated that FOXK1 and FOXK2 promote CM glycolysis,repress oxidative phosphorylation,inhibit DNA damage response pathway,leading to increased CM proliferation and heart regeneration after MI in adult mice.On the other hand,FOXK1 can directly bind to the promoter of Ccnb1 to promote its expression and then promote the proliferation of CMs;FOXK2 can directly bind to the promoter of Cdk1 to promote its expression and then promote the proliferation of CMs.This study suggests that FOXK1 and FOXK2 have the potential to become new targets for promoting cardiac repair after MI and treating heart failure.