Korean J Physiol Pharmacol.  2022 Sep;26(5):357-365. 10.4196/kjpp.2022.26.5.357.

Mitochondrial energy metabolic transcriptome profiles during cardiac differentiation from mouse and human pluripotent stem cells

• Cho SW
• Kim HK
• Sung JH
• Kim Y
• Kim JH
• Han J

• KMID: 2532765
• DOI: http://doi.org/10.4196/kjpp.2022.26.5.357

Abstract

Simultaneous myofibril and mitochondrial development is crucial for the cardiac differentiation of pluripotent stem cells (PSCs). Specifically, mitochondrial energy metabolism (MEM) development in cardiomyocytes is essential for the beating function. Although previous studies have reported that MEM is correlated with cardiac differentiation, the process and timing of MEM regulation for cardiac differentiation remain poorly understood. Here, we performed transcriptome analysis of cells at specific stages of cardiac differentiation from mouse embryonic stem cells (mESCs) and human induced PSCs (hiPSCs). We selected MEM genes strongly upregulated at cardiac lineage commitment and in a time-dependent manner during cardiac maturation and identified the protein-protein interaction networks. Notably, MEM proteins were found to interact closely with cardiac maturation-related proteins rather than with cardiac lineage commitment-related proteins. Furthermore, MEM proteins were found to primarily interact with cardiac muscle contractile proteins rather than with cardiac transcription factors. We identified several candidate MEM regulatory genes involved in cardiac lineage commitment (Cck, Bdnf, Fabp4, Cebpα, and Cdkn2a in mESC-derived cells, and CCK and NOS3 in hiPSC-derived cells) and cardiac maturation (Ppargc1α, Pgam2, Cox6a2, and Fabp3 in mESC-derived cells, and PGAM2 and SLC25A4 in hiPSC-derived cells). Therefore, our findings show the importance of MEM in cardiac maturation.

Keyword

Cardiac myocytes; Mitochondria; Pluripotent stem cell

Figure

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  Fig. 1 Significantly upregulated MEM-related genes at cardiac lineage commitment and during cardiac maturation. (A) Sampling time points of mESC-derived cells: Flk1+ MPCs on day 4.5, PDGFRα+ CLCs on day 6, and αMHC+ cardiomyocytes on day 10.5. (B) Sampling time points of hiPSC-derived cells: KDR+PDGFRα+ cells on days 4 and 5 and cardiomyocytes on day 19. (C) Significantly upregulated MEM genes in mESC-derived PDGFRα+ CLCs at cardiac lineage commitment. (D) MEM genes with continuous time-dependent upregulation during cardiac maturation in mESC-derived cells. (E) Significantly upregulated MEM genes in hiPSC-derived KDR+PDGFRα+ CPCs at cardiac lineage commitment. (F) MEM genes undergoing continuous time-dependent upregulation during cardiac maturation in hiPSC-derived cells. αMHC, alpha-myosin heavy chain; CLC, cardiac lineage-committed cell; CPC, cardiac progenitor cell; CM, cardiomyocyte; Flk1, vascular endothelial growth factor receptor 2; hiPSC, human induced pluripotent stem cell; KDR, kinase insert domain receptor; MEM, mitochondrial energy metabolism; mESC, mouse embryonic stem cell; MPC, mesodermal precursor cell; PDGFRα, platelet-derived growth factor receptor alpha.

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  Fig. 2 Protein-protein interaction networks from mESC-derived cells. (A) PPI networks of MEM and cardiac lineage commitment and maturation in mESC-derived cells. (B) PPI network connection of MEM regulatory genes and cardiac lineage commitment-related genes in mESC-derived cells. (C) PPI network connection of MEM regulatory genes and cardiac maturation-related genes in mESC-derived cells. The red and blue colors mean the gene expression changes at the stages of cardiac maturation and lineage commitment. The circle means cardiac lineage commitment related gene, the triangle means cardiac maturation related gene, and the diamond is mitochondria related gene. The gray line is the interactions among the genes except interactions of mitochondria-cardiac lineage commitment related genes (green line) or mitochondria-cardiac maturation related genes (red line). mESC, mouse embryonic stem cell; PPI, protein-protein interaction; MEM, mitochondrial energy metabolism.

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  Fig. 3 Protein-protein interaction networks from hiPSC-derived cells. (A) PPI networks of MEM and cardiac lineage commitment and maturation in hiPSC-derived cells. (B) PPI network connection of MEM regulatory genes and cardiac lineage commitment-related genes in hiPSC-derived cells. (C) PPI network connection of MEM regulatory genes and cardiac maturation-related genes in hiPSC-derived cells. The red and blue colors mean the gene expression changes at the stages of cardiac maturation and lineage commitment. The circle means cardiac lineage commitment related gene, the triangle means cardiac maturation related gene, and the diamond is mitochondria related gene. The gray line is the interactions among the genes except interactions of mitochondria-cardiac lineage commitment related genes (green line) or mitochondria-cardiac maturation related genes (red line). hiPSC, human induced pluripotent stem cell; PPI, protein-protein interaction; MEM, mitochondrial energy metabolism.

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  Fig. 4 Confirmation of MEM regulatory gene candidates during cardiac differentiation from hESCs. (A) Schematic cardiomyocyte differentiation protocol and sampling time points of the hESC-derived cells: mesodermal cells on day 3, cardiac progenitor cells on day 8, and cardiomyocytes on day 16, formed from the differentiation of H9 hESCs (scale bars, 50 μm). (B) The expression level of CCK, NOS3, PGAM2, and SLC25A4 at each developmental stage from microarray. (C) The expression level of CCK, NOS3, PGAM2, and SLC25A4 at each developmental stage from qPCR. *p-value < 0.005. RBIn, RPMI1640+B27 without Insulin; RB+, RPMI1640+B27 supplement. MEM, mitochondrial energy metabolism; hESC, human embryonic stem cell; CCK, cholecystokinin; NOS3, nitric oxide synthase 3; SLC25A4, solute carrier family 25 member 4; qPCR, quantitative polymerase chain reaction.

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  Fig. 5 Schematic diagram of MEM regulation at at cardiac lineage commitment and cardiac maturation. MEM, mitochondrial energy metabolism; MPC, mesodermal precursor cell; CLC, cardiac lineage-committed cell; CPC, cardiac progenitor cell; CM, cardiomyocyte.

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