Abstract
The initiation of heartbeat is an essential step in cardiogenesis in the heart primordium, but it remains unclear how intracellular metabolism responds to increased energy demands after heartbeat initiation. In this study, embryos in Wistar rats at embryonic day 10, at which heartbeat begins in rats, were divided into two groups by the heart primordium before and after heartbeat initiation and their metabolic characteristics were assessed. Metabolome analysis revealed that increased levels of ATP, a main product of glucose catabolism, and reduced glutathione, a by-product of the pentose phosphate pathway, were the major determinants in the heart primordium after heartbeat initiation. Glycolytic capacity and ATP synthesis-linked mitochondrial respiration were significantly increased, but subunits in complexes of mitochondrial oxidative phosphorylation were not upregulated in the heart primordium after heartbeat initiation. Hypoxia-inducible factor (HIF)-1α was activated and a glucose transporter and rate-limiting enzymes of the glycolytic and pentose phosphate pathways, which are HIF-1α-downstream targets, were upregulated in the heart primordium after heartbeat initiation. These results suggest that the HIF-1α-mediated enhancement of glycolysis with activation of the pentose phosphate pathway, potentially leading to antioxidant defense and nucleotide biosynthesis, covers the increased energy demand in the beating and developing heart primordium.
Highlights
That mitochondrial oxidative phosphorylation (OXPHOS), which can produce large amounts of ATP compared with those produced by anerobic glycolysis under the condition of a sufficient supply of oxygen and energy substrates, contributes less to energy metabolism in the embryonic heart than that in the matured heart[10,11,12]
We investigated whether hypoxia-inducible factor (HIF)-1α is activated in response to increased energy demand and whether expression of its downstream targets in glycolysis, pentose phosphate pathway, and tricarboxylic acid (TCA) cycle is altered in the heart primordium after heartbeat initiation compared with that before heartbeat initiation
Protein levels of HIF-1β, an obligatory dimerization partner for HIF-1α, and prolyl hydroxylase-1 (PHD1), an oxygen-sensing protein that is not regulated by HIF-1α and controls degradation of α-subunits of HIFs via hydroxylation[27], were comparable in the pre- and post-heartbeat groups, whereas the von Hippel–Lindau protein, a substrate recognition component of an E3-ubiquitin ligase that ubiquitylates HIF-1α, was significantly decreased in the post-heartbeat group compared with that in the pre-heartbeat group (Fig. 4B,C). These findings suggest that HIF-1α is upregulated in the heart primordium after heartbeat initiation with reduced pVHL expression presumably contributing to reduction of HIF-1α ubiquitination and degradation
Summary
That mitochondrial oxidative phosphorylation (OXPHOS), which can produce large amounts of ATP compared with those produced by anerobic glycolysis under the condition of a sufficient supply of oxygen and energy substrates, contributes less to energy metabolism in the embryonic heart than that in the matured heart[10,11,12]. Cristae density and the number of mitochondria in cardiomyocytes have been reported to be smaller in the embryonic heart than in the matured heart[12,13,14], suggesting less reliance on mitochondrial OXPHOS for the production of ATP in cardiac progenitor cells or cardiomyocytes in the embryo It remains unclear whether mitochondria play any roles in response to increased energy demand to initiate excitation–contraction coupling in the developing heart primordium. In association with HIF pathways and energy metabolism, it has been reported that activation of HIF-1α pathways transcriptionally upregulates glucose transporters, rate-limiting enzymes in the glycolytic pathway and the pentose phosphate pathway, and pyruvate dehydrogenase kinase-1 (PDK1), leading to inactivation of pyruvate dehydrogenase (PDH) complex in the tricarboxylic acid (TCA) c ycle[22,23,24] These regulations potentially result in enhanced glycolysis, increased activity of the pentose phosphate pathway, and decreased mitochondrial respiration. We investigated whether HIF-1α is activated in response to increased energy demand and whether expression of its downstream targets in glycolysis, pentose phosphate pathway, and TCA cycle is altered in the heart primordium after heartbeat initiation compared with that before heartbeat initiation
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