Candice Baker

Development of aerobic metabolism in utero: requirement for mitochondrial function during embryonic and foetal periods

Introduction The mammalian embryo requires an increasing amount of energy as it develops during the embryonic and foetal stages. The main production mechanism of adenosine triphosphate (ATP) during the early developmental period (post-implantation to beginning of organogenesis) is via anaerobic glycolysis. Despite the fact that it has long been known that embryonic/foetal development becomes dependent upon oxidative phosphorylation in mitochondria, a common misconception persists in the scientific literature indicating that a shift to aerobic metabolism occurs only after birth (‘foetal-shift’). Biotechnology has facilitated the creation of targeted genetic knockout models in mice, and many of these clearly demonstrate the necessity of mitochondrial function for proper development of the embryo/ foetus in the womb. This review highlights representative examples where the loss of genes influencing mitochondrial structure and function causes severe energy-deficiency and/or mitochondrial dysfunction leading to prenatal lethality. Conclusion Critical examination of the idea that anaerobic metabolism is the primary generator of ATP throughout gestation in mammals suggests that this may not be entirely true since the developmental switch to aerobic metabolism actually occurs well before birth. This earlier ‘embryonic-shift’ in metabolic mechanism is essential for embryonic survival and foetal development that begins during organogenesis and continues throughout the remainder of prenatal development. Introduction The primary source of adenosine triphosphate (ATP) during foetal development is commonly reported to be from glycolysis and lactate production, with a ‘foetal-shift’ occurring at birth where the primary source of ATP production ‘shifts’ to oxidative phosphorylation in the mitochondria1-3. A typical example of this notion from the literature is stated as follows1,3: ‘...metabolic programming often is referred to as a ‘foetal’ shift because the myocardium of the developing embryo relies mostly on glycolysis and lactate metabolism for its ATP production’. It has been known for more than 40 years, however, that mammalian hearts show an increase in the importance of oxidative phosphorylation for ATP production during the embryonic development4-6. Additionally, there is accumulating evidence from targeted genetic studies in mice showing embryonic lethality due to disruption of genes associated with the mitochondria and/or dysfunctional oxidative phosphorylation. Moreover, cardiovascular development has been well studied using transgenic mice demonstrating embryonic lethality occurring around the time of mitochondrial maturation during organogenesis7,8. Organogenesis begins around embryonic day 8.0 (E8.0) in mice, which is equivalent to about day 17–19 (Carnegie Stage 8) in humans9,10. Interestingly, this is approximately the stage of development when increased embryonic lethality due to the genetic disruption of mitochondrial associated genes occurs (Table 1) thereby demonstrating that embryonic mitochondrial function is indeed critical for embryonic and foetal development in utero. The aim of this review was to discuss the requirement for mitochondrial function during embryonic and foetal periods in the development of aerobic metabolism in utero. Genetic knockouts of mitochondrial DNA-associated genes Mitochondria are unique organelles as they contain their own DNA (mtDNA) encoding for a set of 37 mitochondrial genes. These genes encode for a variety of proteins including ribosomal RNAs, transfer RNAs and 13 subunits of the electron transport chain11. Although mitochondria contain their own DNA, the majority of the proteins found within the mitochondria are encoded by nuclear DNA. Nuclear DNA encodes all proteins required for mtDNA synthesis. The only DNA polymerase found within mitochondria, DNA polymerase gamma (Polg) is thought to be solely responsible for the replication and repair of mtDNA12. Mutations of the mouse Pol I-like catalytic core (PolgA) gene resulted in an embryonic lethal model that died between E7.5–E8.5 with severe mtDNA deletions13. Additionally, this model demonstrated a respiratory chain dysfunction by a lack of cytochrome c oxidase (COX) staining13. A similar model with the disruption of a Polg accessory subunit, Polg2, resulted in lethality between E8.0–E8.5 due to defective * Corresponding author Email: steven.ebert@ucf.edu 1 Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida 32827, USA Ph ys io lo gy & B io ch em is tr y

Physiological and genomic consequences of adrenergic deficiency during embryonic/fetal development in mice: impact on retinoic acid metabolism

Adrenergic hormones are essential for early heart development. To gain insight into understanding how these hormones influence heart development, we evaluated genomic expression changes in embryonic hearts from adrenergic-deficient and wild-type control mice. To perform this study, we used a mouse model with targeted disruption of the Dopamine β-hydroxylase (Dbh) gene, whose product is responsible for enzymatic conversion of dopamine into norepinephrine. Embryos homozygous for the null allele (Dbh(-/-)) die from heart failure beginning as early as embryonic day 10.5 (E10.5). To assess underlying causes of heart failure, we isolated hearts from Dbh(-/-) and Dbh(+/+) embryos prior to manifestation of the phenotype and examined gene expression changes using genomic Affymetrix 430A 2.0 arrays, which enabled simultaneous evaluation of >22,000 genes. We found that only 22 expressed genes showed a significant twofold or greater change, representing ~0.1% of the total genes analyzed. More than half of these genes are associated with either metabolism (31%) or signal transduction (22%). Remarkably, several of the altered genes encode for proteins that are directly involved in retinoic acid (RA) biosynthesis and transport. Subsequent evaluation showed that RA concentrations were significantly elevated by an average of ~3-fold in adrenergic-deficient (Dbh(-/-)) embryos compared with controls, thereby suggesting that RA may be an important downstream mediator of adrenergic action during embryonic heart development.

Distinctive Left-Sided Distribution of Adrenergic-Derived Cells in the Adult Mouse Heart

Adrenaline and noradrenaline are produced within the heart from neuronal and non-neuronal sources. These adrenergic hormones have profound effects on cardiovascular development and function, yet relatively little information is available about the specific tissue distribution of adrenergic cells within the adult heart. The purpose of the present study was to define the anatomical localization of cells derived from an adrenergic lineage within the adult heart. To accomplish this, we performed genetic fate-mapping experiments where mice with the cre-recombinase (Cre) gene inserted into the phenylethanolamine-n-methyltransferase (Pnmt) locus were cross-mated with homozygous Rosa26 reporter (R26R) mice. Because Pnmt serves as a marker gene for adrenergic cells, offspring from these matings express the β-galactosidase (βGAL) reporter gene in cells of an adrenergic lineage. βGAL expression was found throughout the adult mouse heart, but was predominantly (89%) located in the left atrium (LA) and ventricle (LV) (p<0.001 compared to RA and RV), where many of these cells appeared to have cardiomyocyte-like morphological and structural characteristics. The staining pattern in the LA was diffuse, but the LV free wall displayed intermittent non-random staining that extended from the apex to the base of the heart, including heavy staining of the anterior papillary muscle along its perimeter. Three-dimensional computer-aided reconstruction of XGAL+ staining revealed distribution throughout the LA and LV, with specific finger-like projections apparent near the mid and apical regions of the LV free wall. These data indicate that adrenergic-derived cells display distinctive left-sided distribution patterns in the adult mouse heart.

Impaired cardiac energy metabolism in embryos lacking adrenergic stimulation

As development proceeds from the embryonic to fetal stages, cardiac energy demands increase substantially, and oxidative phosphorylation of ADP to ATP in mitochondria becomes vital. Relatively little, however, is known about the signaling mechanisms regulating the transition from anaerobic to aerobic metabolism that occurs during the embryonic period. The main objective of this study was to test the hypothesis that adrenergic hormones provide critical stimulation of energy metabolism during embryonic/fetal development. We examined ATP and ADP concentrations in mouse embryos lacking adrenergic hormones due to targeted disruption of the essential dopamine β-hydroxylase (Dbh) gene. Embryonic ATP concentrations decreased dramatically, whereas ADP concentrations rose such that the ATP/ADP ratio in the adrenergic-deficient group was nearly 50-fold less than that found in littermate controls by embryonic day 11.5. We also found that cardiac extracellular acidification and oxygen consumption rates were significantly decreased, and mitochondria were significantly larger and more branched in adrenergic-deficient hearts. Notably, however, the mitochondria were intact with well-formed cristae, and there was no significant difference observed in mitochondrial membrane potential. Maternal administration of the adrenergic receptor agonists isoproterenol or l-phenylephrine significantly ameliorated the decreases in ATP observed in Dbh-/- embryos, suggesting that α- and β-adrenergic receptors were effective modulators of ATP concentrations in mouse embryos in vivo. These data demonstrate that adrenergic hormones stimulate cardiac energy metabolism during a critical period of embryonic development.

Targeting of the Enhanced Green Fluorescent Protein Reporter to Adrenergic Cells in Mice

Adrenaline and noradrenaline are important neurotransmitter hormones that mediate physiological stress responses in adult mammals, and are essential for cardiovascular function during a critical period of embryonic/fetal development. In this study, we describe a novel mouse model system for identifying and characterizing adrenergic cells. Specifically, we generated a reporter mouse strain in which a nuclear-localized enhanced green fluorescent protein gene (nEGFP) was inserted into exon 1 of the gene encoding Phenylethanolamine n-methyltransferase (Pnmt), the enzyme responsible for production of adrenaline from noradrenaline. Our analysis demonstrates that this knock-in mutation effectively marks adrenergic cells in embryonic and adult mice. We see expression of nEGFP in Pnmt-expressing cells of the adrenal medulla in adult animals. We also note that nEGFP expression recapitulates the restricted expression of Pnmt in the embryonic heart. Finally, we show that nEGFP and Pnmt expressions are each induced in parallel during the in vitro differentiation of pluripotent mouse embryonic stem cells into beating cardiomyocytes. Thus, this new mouse genetic model should be useful for the identification and functional characterization of adrenergic cells in vitro and in vivo.