Investigating the role of mitophagy on the maturation of pluripotent stem cell-derived cardiomyocytes

Abstract

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) hold significant potential for regenerative therapies and drug screening.However, the immature status of PSC-CMs hinders its clinical utilisation. Thus, it is of great importance to improve PSC-CM maturation. Multifaceted methods, including hormonal induction, prolonged culture and tissue engineering have been used to enhance the maturity of PSC-CMs, but a comprehensive maturation status has yet to be achieved. The perinatal phase has been recognised as a critical window for the in vivo maturation of mammalian CMs.

Shortly after birth, CMs undergo a transient period of starvation which leads to autophagy and mitophagy, ultimately resulting in the structural and metabolic alterations of CMs. Mitophagy is the autophagic removal of mitochondria and can be divided into ubiquitin-dependent and ubiquitin-independent pathways. Ubiquitin-independent mitophagy involves mitophagy receptors like BNIP3 and FUNDC1 which interact directly with LC3 on autophagosomes. PINK1-Parkin are responsible for ubiquitin-mediated mitophagy by tagging ubiquitin to mitochondrial membrane proteins as an autophagic signal. Notably, Parkin has been shown to be associated with the maturation of CMs.

In the current study, nutrient depletion and bafilomycin A1 (Baf A1) treatment were used to promote and inhibit mitophagy in PSC-CMs, respectively. Interestingly, an increase in mitophagy in PSC-CMs was accompanied with enhanced maturation as reflected by various parameters, including an increase in calcium transient kinetics, cell size and sarcomere length. On the other hand, Baf A1-treated PSC-CMs exhibited decreased maturation in various aspects such as reduced calcium handling capability, decreased mitochondrial content and decreased percentage of cells with organized sarcomere.

These data suggest that mitophagy positively regulates PSC-CM maturation. In addition, expression of mitophagy-related genes was profiled across different maturation timepoints of PSC-CMs.

Our data showed that the gene expression of BNIP3 increased significantly as PSC-CMs mature. Interestingly, expression of BNIP3 was found to be increased immediately after starvation treatment. Therefore, it is tempting to ask whether receptor-mediated mitophagy, especially BNIP3-mediated mitophagy, would contribute to PSC-CM maturation. By understanding how mitophagy contributes to the maturation of CMs and its underlying molecular mechanism, this study aims to delineate an understudied pathway that holds great promise to enhance PSC-CM maturity.