Glycolytic network restructuring integral to the energetics of embryonic stem cell cardiac differentiation

Abstract

Decoding of the bioenergetic signature underlying embryonic stem cell cardiac differentiation has revealed a mandatory transformation of the metabolic infrastructure with prominent mitochondrial network expansion and a distinctive switch from glycolysis to oxidative phosphorylation. Here, we demonstrate that despite reduction in total glycolytic capacity, stem cell cardiogenesis engages a significant transcriptome, proteome, as well as enzymatic and topological rearrangement in the proximal, medial, and distal modules of the glycolytic pathway. Glycolytic restructuring was manifested by a shift in hexokinase (Hk) isoforms from Hk-2 to cardiac Hk-1, with intracellular and intermyofibrillar localization mapping mitochondrial network arrangement. Moreover, upregulation of cardiac-specific enolase 3, phosphofructokinase, and phosphoglucomutase and a marked increase in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) phosphotransfer activity, along with apparent post-translational modifications of GAPDH and phosphoglycerate kinase, were all distinctive for derived cardiomyocytes compared to the embryonic stem cell source. Lactate dehydrogenase (LDH) isoforms evolved towards LDH-2 and LDH-3, containing higher proportions of heart-specific subunits, and pyruvate dehydrogenase isoforms rearranged between E1alpha and E1beta, transitions favorable for substrate oxidation in mitochondria. Concomitantly, transcript levels of fetal pyruvate kinase isoform M2, aldolase 3, and transketolase, which shunt the glycolytic with pentose phosphate pathways, were reduced. Collectively, changes in glycolytic pathway modules indicate active redeployment, which would facilitate connectivity of the expanding mitochondrial network with ATP utilization sites. Thus, the delineated developmental dynamics of the glycolytic phosphotransfer network is integral to the remodeling of cellular energetic infrastructure underlying stem cell cardiogenesis.

Keywords: bioenergetics; cardiogenesis; embryonic stem cells; proteomics; transcriptome.

Figure 1. Embryonic stem cell cardiac differentiation is associated with enhanced mitochondrial and electrical activities, reduced glycolytic capacity and restructuring of the glycolytic transcriptome

(A) Cardiomyocytes within an embryoid body’s beating area at different stages (I–III) of formation contain greater numbers of mitochondria giving off a stronger JC-1 signal (green) and have a higher membrane potential (RH237 signal, red) than the surrounding cells; (B) Glycolytic capacity in uncoupled (DNP, 50 μM) and uncoupled-respiration inhibited (DNP/KCN, 50 μM and 0.5 mM, respectively) states is lower in ES cell-derived cardiomyocytes (CM) compared to ES cells (ES); (C) Microarray analyses of ES cells and cardiomyocytes indicate downregulated and upregulated transcripts of enzymes in the glycolytic pathway. Scale bars indicate 10 μm (I and II panels) and 20 μm (III panel).

Figure 2. Transcriptomic and topological restructuring of the entry step in the glycolytic pathway: increase connection with mitochondria

(A) A shift in hexokinase isoforms from Hxk-2 towards Hxk-1 and marginal reduction in enzyme activity in ES cell-derived cardiomyocytes (CM). Immunocytochemistry indicates that, compared to (B) ES cells, (C) cardiomyocytes have increased Hxk-1 abundance with a stippled pattern of intracellular localization and higher perinuclear concentration corresponding to mitochondrial network arrangement; the myofibrillar mesh is stained with α-actinin (red). Scale bar indicates 10 μm.

Figure 3. Transcriptomic, proteomic and enzymatic restructuring of the middle segment of the glycolytic pathway

(A) Embryonic stem (ES) cell cardiac differentiation is associated with increased GAPDH phosphotransfer activity, without parallel mRNA or protein changes, although there is a modest increase in post-translational modification of GAPDH in ES cell-derived cardiomyocytes (CM) detected by 2-D gels, densitometry and MS/MS analysis. (B) Downregulation of messenger RNA levels of PGK1 and total PGK activity with increased post-translational modifications of protein detected by 2-D gels, densitometry and MS/MS analysis during ES cell cardiac differentiation.

Figure 4. Transcriptomic, proteomic and enzymatic restructuring of the ending segment of the glycolytic pathway

(A) Zymogram (left) analysis by densitometry (right) indicates a shift in LDH isoforms towards cardiac LDH-2 and LDH-3 in ES cell-derived cardiomyocytes. (B) LDH mRNA levels and (C) activity in ES cells and cardiomyocytes. (D) Proteomic analysis of LDH-M in ES cells and cardiomyocytes.

Figure 5. A shift in pyruvate dehydrogenase (PDH) isoforms and downregulation of fetal pyruvate kinase PK-m2 and transketolase Tkt transcripts during ES cell cardiac differentiation

(A and B) Differential regulation of PDH isoform transcriptome and proteome during ES cell cardiogenesis. (C) Reduced fetal PK-m2 and (B) Tkt transcript levels in ES cell-derived cardiomyocytes.

Figure 6. Developmental restructuring and intracellular positioning of glycolytic phosphotransfer enzymes facilitate integration of mitochondrial energetics with ATP utilization sites

Schematic representation of the glycolytic pathway transformed into a network to assume a new energetic function of high-energy phosphotransfer from mitochondria to cellular ATPases. Marked in cyan color are components undergoing transcriptome, proteome or enzymatic activity restructuring during stem cell cardiogenesis. PPP – pentose phosphate pathway; ANT – adenine nucleotide translocator; i.m. and o.m. – inner and outer mitochondrial membranes, respectively.