Abstract Body: Background: The elevated level of reactive oxygen species (ROS) has been reported to drive the cell-cycle-exit of neonatal mouse cardiomyocytes and impede their regeneration. On the contrary, the roles of reductive stress in perinatal development and regeneration of cardiomyocytes are still poorly understood. To counterbalance increased oxidative stress, antioxidant enzymes are expressed to remove excessive ROS. Our previous work uncovered a role of the transcription factor Pitx2 as a redox regulator in the ventricular myocardium. Pitx2 protects myocardium by positively regulating genes that encode antioxidant scavengers. In regenerating neonatal hearts, Pitx2 expression is activated by Nrf2, a known antioxidant regulator. Under oxidative stress, Nrf2 dissociates from the Keap1-Cul3-Rbx1 degradation complex and translocates to the nucleus where it promotes Pitx2 expression. Interestingly, our RNA-Seq and ChIP-Seq studies showed that Pitx2 promotes Rbx1 expression which degrades Nrf2. We hypothesize that this negative feedback loop (Image 1) is required to prevent reductive stress in regenerating cardiomyocytes.
Methods: Cre-Lox models were used to specifically knock out Nrf2 in cardiomyocytes. Constitutively active Nrf2 (caNrf2) was expressed specifically in cardiomyocyte using AAV9. Guide RNAs were delivered into Myh6Cas9 mouse heart using AAV9 to knock out Rbx1 in cardiomyocytes. Neonatal ROS was transiently suppressed using high dose of N-acetyl-cysteine (NAC). Following left anterior descending artery occlusion (LAD-O), cardiac regeneration was assessed by Masson’s trichrome staining and echocardiography. Markers of cell cycle activity and cell death were assessed by immunofluorescence. RNA-Seq was performed on isolated cardiomyocytes from NAC and saline treated hearts after sham or LAD-O procedure.
Results: Pitx2 promotes the degradation of Nrf2 by inducing the expression of Rbx1. In cardiomyocytes, Rbx1, but not Nrf2, is required for neonatal heart regeneration. Meanwhile, expression of caNrf2 in cardiomyocytes increases mortality after LAD-O. Removing ROS in neonatal heart prevents cardiomyocyte maturation and disrupts injury response after LAD-O.
Conclusions: Together, these results suggest that a fine balance between reductive and oxidative activity is required for neonatal heart regeneration and postnatal heart development. Excessive antioxidant activity will cause reductive stress and impede neonatal heart regeneration after MI.