Comparison of DNA methylation patterns across tissue types in infants with tetralogy of Fallot.

Background: Environmental factors may influence the development of tetralogy of Fallot (TOF), and DNA methylation patterns may reveal specific chemical signatures of perturbations during cardiac development. We investigated whether blood and buccal cells could be viable surrogates for myocardium.

Methods: We measured epigenome-wide DNA methylation at 866,895 5'-cytosine-phosphate-guanine-3' (CpG) sites in blood (n=3), buccal cells (n=3), and right ventricular myocardium (n=4) collected from infants with TOF and compared the percent of differentially methylated CpG sites across tissue types.

Maternal binge alcohol consumption leads to distinctive acute perturbations in embryonic cardiac gene expression profiles.

Background: Excessive alcohol consumption during pregnancy is associated with high risk of congenital heart defects, but it is unclear how alcohol specifically affects heart development during the acute aftermath of a maternal binge drinking episode. We hypothesize that administration of a single maternal binge dose of alcohol to pregnant mice at embryonic day 9.5 (E9.

Antiretroviral Drugs Regulate Epigenetic Modification of Cardiac Cells Through Modulation of H3K9 and H3K27 Acetylation.

Antiretroviral therapy (ART) has significantly reduced the rate of mortality in HIV infected population, but people living with HIV (PLWH) show higher rates of cardiovascular disease (CVD). However, the effect of antiretroviral (ARV) drug treatment on cardiac cells is not clear. In this study, we explored the effect of ARV drugs in cardiomyocyte epigenetic remodeling.

Hypothalamic MC4R regulates glucose homeostasis through adrenaline-mediated control of glucose reabsorption via renal GLUT2 in mice.

Aims/hypothesis: Melanocortin 4 receptor (MC4R) mutation is the most common cause of known monogenic obesity in humans. Unexpectedly, humans and rodents with MC4R deficiency do not develop hyperglycaemia despite chronic obesity and insulin resistance. To explain the underlying mechanisms for this phenotype, we determined the role of MC4R in glucose homeostasis in the presence and absence of obesity in mice.

Adrenergic chromaffin cells are adrenergic even in the absence of epinephrine.

Adrenal chromaffin cells release epinephrine (EPI) and norepinephrine (NE) into the bloodstream as part of the homeostatic response to situations like stress. Here we utilized EPI-deficient mice generated by knocking out (KO) the phenylethanolamine N-methyltransferase (Pnmt) gene. These Pnmt-KO mice were bred to homozygosis but displayed no major phenotype.

Pnmt-Derived Cardiomyocytes: Anatomical Localization, Function and Future Perspectives.

In this mini-review, we provide an overview of phenylethanolamine-N-methyl transferase (Pnmt)-derived cardiomyocytes (PdCMs), a recently discovered cardiomyocyte subpopulation. We discuss their anatomical localization, physiological characteristics, possible function, and future perspectives. Their unique distribution in the heart, electrical activity, Ca transient properties, and potential role in localized adrenergic signaling are discussed.

Metabolomics reveals critical adrenergic regulatory checkpoints in glycolysis and pentose-phosphate pathways in embryonic heart.

Cardiac energy demands during early embryonic periods are sufficiently met through glycolysis, but as development proceeds, the 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 ∼10-fold less cardiac ATP compared with littermate controls.

Conditional ablation and conditional rescue models for Casq2 elucidate the role of development and of cell-type specific expression of Casq2 in the CPVT2 phenotype.

Cardiac calsequestrin (Casq2) associates with the ryanodine receptor 2 channel in the junctional sarcoplasmic reticulum to regulate Ca2+ release into the cytoplasm. Patients carrying mutations in CASQ2 display low resting heart rates under basal conditions and stress-induced polymorphic ventricular tachycardia (CPVT). In this study, we generate and characterize novel conditional deletion and conditional rescue mouse models to test the influence of developmental programs on the heart rate and CPVT phenotypes.

Intact calcium signaling in adrenergic-deficient embryonic mouse hearts.

Mouse embryos that lack the ability to produce the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to disruption of the dopamine beta-hydroxylase (Dbh) gene inevitably perish from heart failure during mid-gestation. Since adrenergic stimulation is well-known to enhance calcium signaling in developing as well as adult myocardium, and impairments in calcium signaling are typically associated with heart failure, we hypothesized that adrenergic-deficient embryonic hearts would display deficiencies in cardiac calcium signaling relative to adrenergic-competent controls at a developmental stage immediately preceding the onset of heart failure, which first appears beginning or shortly after mouse embryonic day 10.5 (E10.

Optogenetic Control of Heart Rhythm by Selective Stimulation of Cardiomyocytes Derived from Pnmt Cells in Murine Heart.

In the present study, channelrhodopsin 2 (ChR2) was specifically introduced into murine cells expressing the Phenylethanolamine n-methyltransferase (Pnmt) gene, which encodes for the enzyme responsible for conversion of noradrenaline to adrenaline. The new murine model enabled the identification of a distinctive class of Pnmt-expressing neuroendocrine cells and their descendants (i.e.

Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane.

Vesicle fusion is executed via formation of an Ω-shaped structure (Ω-profile), followed by closure (kiss-and-run) or merging of the Ω-profile into the plasma membrane (full fusion). Although Ω-profile closure limits release but recycles vesicles economically, Ω-profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how Ω-profile merging is mediated is poorly understood in endocrine cells and neurons containing small ∼30-300 nm vesicles.

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.

Attenuated aortic vasodilation and sympathetic prejunctional facilitation in epinephrine-deficient mice: selective impairment of β2-adrenoceptor responses.

It has been suggested that there is a link between epinephrine synthesis and the development of β2-adrenoceptor-mediated effects, but it remains to be determined whether this development is triggered by epinephrine. The aim of this study was to characterize β-adrenoceptor-mediated relaxation and facilitation of norepinephrine release in the aorta of phenylethanolamine-N-methyltransferase-knockout (Pnmt-KO) mice. Catecholamines were quantified by reverse-phase high-performance liquid chromatography-electrochemical detection.

Therapeutic potential of Pnmt+ primer cells for neuro/myocardial regeneration.

Phenylethanolamine n-methyltransferase (Pnmt) catalyzes the conversion of norepinephrine into epinephrine, and thus serves as a marker of adrenergic cells. In adults, adrenergic cells are present in the adrenal medullae and the central and peripheral (sympathetic) nervous systems where they play key roles in stress responses and a variety of other functions. During early embryonic development, however, Pnmt first appears in the heart where it is associated with specialized myocytes in the pacemaking and conduction system.

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.

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.

Adrenergic deficiency leads to impaired electrical conduction and increased arrhythmic potential in the embryonic mouse heart.

To determine if adrenergic hormones play a critical role in the functional development of the cardiac pacemaking and conduction system, we employed a mouse model where adrenergic hormone production was blocked due to targeted disruption of the dopamine β-hydroxylase (Dbh) gene. Immunofluorescent histochemical evaluation of the major gap junction protein, connexin 43, revealed that its expression was substantially decreased in adrenergic-deficient (Dbh-/-) relative to adrenergic-competent (Dbh+/+ and Dbh+/-) mouse hearts at embryonic day 10.5 (E10.

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.

Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques.

Background: Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun") delivery to target tissues have not been as extensively explored.

Reduction of sympathetic activity via adrenal-targeted GRK2 gene deletion attenuates heart failure progression and improves cardiac function after myocardial infarction.

Chronic heart failure (HF) is characterized by sympathetic overactivity and enhanced circulating catecholamines (CAs), which significantly increase HF morbidity and mortality. We recently reported that adrenal G protein-coupled receptor kinase 2 (GRK2) is up-regulated in chronic HF, leading to enhanced CA release via desensitization/down-regulation of the chromaffin cell alpha(2)-adrenergic receptors that normally inhibit CA secretion. We also showed that adrenal GRK2 inhibition decreases circulating CAs and improves cardiac inotropic reserve and function.

Generation of novel reporter stem cells and their application for molecular imaging of cardiac-differentiated stem cells in vivo.

Stem cell therapies offer the potential for repair and regeneration of cardiac tissue. To facilitate evaluation of stem cell activity in vivo, we created novel dual-reporter mouse embryonic stem (mES) cell lines that express the firefly luciferase (LUC) reporter gene under the control of the cardiac sodium-calcium exchanger-1 (Ncx-1) promoter in the background of the 7AC5-EYFP mES cell line that constitutively expresses the enhanced yellow fluorescent protein (EYFP). We compared the ability of recombinant clonal cell lines to express LUC before and after induction of cardiac differentiation in vitro.

Catecholamine-synthesizing cells in the embryonic mouse heart.

The heart is a primary source of epinephrine and norepinephrine during embryonic development, yet little is known about the cardiac cells that produce these catecholamine hormones. To identify when and where catecholamine-synthesizing cells are found in the embryonic heart, we developed a novel mouse genetic model by "knocking-in" the Cre-recombinase gene to the locus encoding for the epinephrine biosynthetic enzyme, phenylethanolamine n-methyltransferase. When crossed with ROSA26 reporter mice, the beta-galactosidase gene is activated in adrenergic cells.

Noninvasive tracking of cardiac embryonic stem cells in vivo using magnetic resonance imaging techniques.

Despite rapid advances in the stem cell field, the ability to identify and track transplanted or migrating stem cells in vivo is limited. To overcome this limitation, we used magnetic resonance imaging (MRI) to detect and follow transplanted stem cells over a period of 28 days in mice using an established myocardial infarction model. Pluripotent mouse embryonic stem (mES) cells were expanded and induced to differentiate into beating cardiomyocytes in vitro.

Kcnq1 contributes to an adrenergic-sensitive steady-state K+ current in mouse heart.

It has been suggested that Kcne1 subunits are required for adrenergic regulation of Kcnq1 potassium channels. However, in adult mouse hearts, which do not express Kcne1, loss of Kcnq1 causes a Long QT phenotype during adrenergic challenge, raising the possibility that native Kcnq1 currents exist and are adrenergically regulated even in absence of Kcne1. Here, we used immunoblotting and immunohistochemical staining to show that Kcnq1 protein is present in adult mouse hearts.