Cardiac energy demands during early embryonic periods are sufficiently met through glycolysis, but as development proceeds, oxidative phosphorylation in mitochondria becomes increasingly vital. Adrenergic hormones are known to stimulate metabolism in adult mammals and are essential for embryonic development, but relatively little is known about their effects on metabolism in the embryonic heart. Here, we show that embryos lacking adrenergic stimulation have approximately 10-fold less cardiac ATP compared to littermate controls. Despite this deficit in steady-state ATP, neither the rates of ATP formation or degradation were affected in adrenergic-deficient hearts, suggesting that ATP synthesis and hydrolysis mechanisms were fully operational. We thus hypothesized that adrenergic hormones stimulate metabolism of glucose to provide chemical substrates for oxidation in mitochondria. To test this hypothesis, we employed a metabolomics-based approach using liquid chromatography/mass spectrometry (LC/MS). Our results showed glucose-1-phosphate and glucose-6-phosphate concentrations were not significantly altered, but several downstream metabolites in both glycolytic and pentosephosphate pathways were significantly lower compared to controls. Further, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) as key enzymes in those respective metabolic pathways whose activity was significantly (p < 0.05) and substantially (80% and 40%, respectively) lower in adrenergic-deficient hearts.Addition of pyruvate and to a lesser extent, ribose, led to significant recovery of steady-state ATP concentrations. These results demonstrate that without adrenergic stimulation, glucose metabolism in the embryonic heart is severely impaired in multiple pathways, ultimately leading to insufficient metabolic substrate availability for successful transition to aerobic respiration needed for survival.
Cardiac energy demands increase during embryonic development, requiring activation of oxidative phosphorylation to convert ADP to ATP in mitochondria. We have recently shown that adrenergic hormones are required to maintain sufficient cardiac energy metabolism during embryonic development, but the specific mechanism(s) underlying this regulation are not known. Mouse embryos lacking the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to targeted loss of the dopamine β-hydroxylase ( Dbh ) gene, have markedly (>50-fold) decreased steady-state ATP/ADP ratios. Rates of ATP synthesis and hydrolysis did not differ between adrenergic-deficient and competent embryos suggesting the enzymatic machinery required for ATP production/consumption is functional. We hypothesized that adrenergic-deficient embryonic hearts are metabolically starved of nutrients leading to energy depletion. To identify changes in metabolism in adrenergic-deficient hearts, we performed LC-MS metabolomics, which showed decreases in all nucleotide triphosphates (NTPs) and NAD(H) confirming energy depletion. Additionally, products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G-6-PDH), and pyruvate dehydrogenase (PDH) were significantly diminished compared to controls, suggesting impaired activity. Enzymatic activities of GAPDH, G-6-PDH, PDH, and glycogen phosphorylase (GP); a well-known enzyme under adrenergic regulation, were measured from the rate of NAD(P)H production. GAPDH, G-6-PDH, and GP activities were significantly decreased (~80%, 40%, and 70% reduction, respectively) compared to controls. Interestingly, GAPDH, G-6-PDH, and GP protein levels, examined by western blot, did not differ from adrenergic-competent controls, thereby suggesting that adrenergic hormones regulate posttranslational activity of these enzymes. These results indicate that mitochondria are metabolically starved due to impairments in glycogenolysis, glycolysis, and pentose phosphate pathways. These findings reveal new mechanistic insights into global adrenergic regulation of major metabolic pathways during embryonic heart development.
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