# TCA cycle intermediates as an adjunct strategy for human iPSC-derived cardiomyocyte maturation

## TL;DR

Adding TCA cycle intermediates helps improve the maturity of heart cells made from stem cells, which could aid in disease modeling and regenerative medicine.

## Contribution

The study shows that supplementing TCA cycle intermediates enhances the maturation of iPSC-derived cardiomyocytes.

## Key findings

- TCA cycle intermediates like succinate and malate improve calcium handling and cellular morphology in iPSC-CM.
- Supplementation enhances electrical activity and mitochondrial health, shifting energy metabolism toward oxidative phosphorylation.
- TCA supplementation upregulates maturation genes and downregulates fetal genes in iPSC-CM.

## Abstract

•Targeted metabolomics demonstrate that TCA cycle intermediates play an important role the maturation of iPSC derived cardiomyocytes.•Supplementation of succinate, malate, fumarate and α-ketoglutarate promotes metabolic and functional maturation of iPSC derived cardiomyocytes.

Targeted metabolomics demonstrate that TCA cycle intermediates play an important role the maturation of iPSC derived cardiomyocytes.

Supplementation of succinate, malate, fumarate and α-ketoglutarate promotes metabolic and functional maturation of iPSC derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are a valuable tool for modeling cardiac diseases, drug testing, and regenerative applications. However, their application is limited by the immature phenotype of iPSC-CM. During maturation from fetal to adult phenotype cardiomyocytes undergo a transition from glycolysis to oxidative phosphorylation for energy production, which is supported by efficient tricarboxylic acid (TCA) cycle activity. Our metabolomics data suggest that the level of intermediates of TCA cycle including succinate, malate, fumarate, and α-ketoglutarate was very low in iPSC-CM. Therefore, we investigated the effect of supplementation with these metabolites on the maturation of iPSC-CM. We cultured iPSC-CM in glucose (Glu), galactose (Gal), or galactose plus TCA cycle intermediates (Gal+TCA) and evaluated the incremental effect of TCA cycle intermediates supplementation relative to Glu and Gal. The treatment with these TCA cycle intermediates led to improved calcium handling and cellular morphology of iPSC-CM relative to Glu and Gal. Furthermore, the treatment with TCA cycle metabolites enhanced electrical activity, improved mitochondrial health, and the cells were shifting toward oxidative phosphorylation relative to Glu only. This shift in the energy metabolism was associated with an upregulation in the expression of cardiomyocyte maturation genes and downregulation in the expression of fetal genes in Gal + TCA group relative to Glu. Overall, the benefits of Gal+TCA supplementation were quite evident compared to Glu alone but generally modest relative to Gal supplementation, supporting that TCA cycle intermediates supplementation can be used as an adjunct strategy to promote iPSC-CM maturation.