Ralf Gilsbach

Comparison of in vitro and in vivo reference genes for internal standardization of real-time PCR data

Real-time PCR is a powerful technique for gene expression studies, which have become increasingly important in a large number of clinical and scientific fields. The significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. The most widely used approach is to use endogenous reference genes (housekeeping genes) as internal standards. This approach is controversially discussed in the literature because none of the reference genes is stably expressed throughout all biological samples. Therefore, candidate reference genes have to be validated for each experimental condition. In our studies, we introduced and evaluated an in vitro synthesized reference cRNA for internal standardization of relative messenger RNA (mRNA) expression patterns. This reference, consisting of the in vitro transcribed coding sequence of aequorin, a jellyfish protein, was incorporated in the extracted RNA. The experimental significance of this approach was representatively tested for the expression of the neurotrophin-3 mRNA in distinct regions of mouse brains. A comparison to three stably expressed reference genes [beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine phosphoribosyl-transferase 1 (HPRT1)] gave evidence that the spiking of template RNA with in vitro transcribed cRNA is a valuable tool for internal standardization of real-time PCR experiments.

Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease

The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity.

α2-Adrenoceptor subtypes—Unexpected functions for receptors and ligands derived from gene-targeted mouse models

Alpha2-adrenoceptors belong to the group of nine adrenoceptors which mediate the biological actions of the endogenous catecholamines adrenaline and noradrenaline. Studies with gene-targeted mice carrying deletions in the genes encoding alpha2A-, alpha2B- or alpha2C-adrenoceptors have provided new insight into adrenergic receptor biology: (1) In principle, all three alpha2-receptor subtypes may operate as presynaptic inhibitory feedback receptors to control the release of noradrenaline and adrenaline or other transmitters from neurons. (2) Pharmacological effects of non-selective alpha2-ligands could be assigned to specific receptor subtypes, e.g. hypotension, sedation and analgesia are mediated via alpha2A-receptors. (3) Alpha2-adrenoceptor deficient mice have helped to uncover novel and unexpected functions of these receptor, e.g. feedback control of catecholamine release via alpha2C-receptors in adrenal chromaffin cells and control of angiogenesis during embryonic development. (4) Additional pharmacological targets for alpha2-adrenoceptor ligands were identified, e.g. inhibition of cardiac HCN2 and HCN4 pacemaker channels by clonidine.

Ablation of Mineralocorticoid Receptors in Myocytes But Not in Fibroblasts Preserves Cardiac Function

Antagonists of the mineralocorticoid receptor improve morbidity and mortality in patients with severe heart failure. However, the cell types involved in these beneficial effects are only partially known. The aim of this work was to evaluate whether genetic deletion of mineralocorticoid receptors in mouse cardiomyocytes or fibroblasts in vivo is cardioprotective after chronic left ventricular pressure overload. After transverse aortic constriction, mice deficient in myocyte mineralocorticoid receptors but not those deficient in fibroblast mineralocorticoid receptors were protected from left ventricular dilatation and dysfunction. After pressure overload, left ventricular ejection fraction was significantly higher in mice lacking myocyte mineralocorticoid receptors (70.2±4.4%) as compared with control mice (54.3±2.5%; P<0.01). Myocyte mineralocorticoid receptor-deficient mice showed mild cardiac hypertrophy at baseline, contributing to reduced left ventricular wall tension at baseline and after pressure overload. Cardiac levels of phospho-extracellular signal–regulated kinase 1/2 were higher in myocyte mineralocorticoid receptor-deficient mice than in control mice after pressure overload. Neither fibroblast nor myocyte mineralocorticoid receptor ablation altered the development of cardiac hypertrophy or fibrosis after pressure overload. Both mineralocorticoid receptor mutant mouse strains developed similar degrees of myocyte apoptosis, proinflammatory gene expression, and macrophage infiltration after pressure overload. Thus, mineralocorticoid receptors in cardiac myocytes but not in fibroblasts protect from cardiac dilatation and failure after chronic pressure overload.

Genetic Dissection of α2-Adrenoceptor Functions in Adrenergic versus Nonadrenergic Cells

α2-Adrenoceptors mediate diverse functions of the sympathetic system and are targets for the treatment of cardiovascular disease, depression, pain, glaucoma, and sympathetic activation during opioid withdrawal. To determine whether α2-adrenoceptors on adrenergic neurons or α2-adrenoceptors on nonadrenergic neurons mediate the physiological and pharmacological responses of α2-agonists, we used the dopamine β-hydroxylase (Dbh) promoter to drive expression of α2A-adrenoceptors exclusively in noradrenergic and adrenergic cells of transgenic mice. Dbh-α2A transgenic mice were crossed with double knockout mice lacking both α2A- and α2C-receptors to generate lines with selective expression of α2A-autoreceptors in adrenergic cells. These mice were subjected to a comprehensive phenotype analysis and compared with wild-type mice, which express α2A- and α2C-receptors in both adrenergic and nonadrenergic cells, and α2A/α2C double-knockout mice, which do not express these receptors in any cell type. We were surprised to find that only a few functions previously ascribed to α2-adrenoceptors were mediated by receptors on adrenergic neurons, including feedback inhibition of norepinephrine release from sympathetic nerves and spontaneous locomotor activity. Other agonist effects, including analgesia, hypothermia, sedation, and anesthetic-sparing, were mediated by α2-receptors in nonadrenergic cells. In dopamine β-hydroxylase knockout mice lacking norepinephrine, the α2-agonist medetomidine still induced a loss of the righting reflex, confirming that the sedative effect of α2-adrenoceptor stimulation is not mediated via autoreceptor-mediated inhibition of norepinephrine release. The present study paves the way for a revision of the current view of the α2-adrenergic receptors, and it provides important new considerations for future drug development.

Increased Expression of the Auxiliary β2-subunit of Ventricular L-type Ca2+ Channels Leads to Single-Channel Activity Characteristic of Heart Failure

Background Increased activity of single ventricular L-type Ca2+-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary β-subunits as a possible explanation. Methods and Results By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC β-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac β-subunits: Unlike β1 or β3 isoforms, β2a and β2b induce a high-activity channel behavior typical of failing myocytes. In accordance, β2-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac CaV1.2 also reveal increased single-channel activity and sarcolemmal β2 expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing (“Adaptive Phase”), reveal the opposite phenotype, viz : reduced single-channel activity accompanied by lowered β2 expression. Additional evidence for the cause-effect relationship between β2-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive CaV1.2 and inducible β2 cardiac overexpression. Here in non-failing hearts induction of β2-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. Conclusions Our study presents evidence of the pathobiochemical relevance of β2-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

Are the pharmacology and physiology of α2adrenoceptors determined by α2‐heteroreceptors and autoreceptors respectively?

α2‐Adrenoceptors are important mediators of physiological responses to the endogenous catecholamines noradrenaline and adrenaline. In addition, α2‐adrenoceptors are pharmacological targets for the treatment of hypertension, sympathetic overactivity and glaucoma. α2‐Adrenoceptors are also targeted to induce sedation and analgesia in anaesthesia and intensive care. α2‐Adrenoceptors were first described as presynaptic receptors inhibiting the release of various transmitters from neurons in the central and peripheral nervous systems. In addition to these presynaptic neuronal receptors, α2‐adrenoceptors were also identified in many non‐neuronal cell types of the body. Gene‐targeting in mice provided a comprehensive assignment of the physiological and pharmacological functions of these receptors to specific α2A‐, α2B‐ and α2C‐adrenoceptor subtypes. However, the specific cell types and signalling pathways involved in these subtype‐specific α2‐adrenoceptor functions were largely unexplored until recently. This review summarizes recent findings from transgenic mouse models, which were generated to define the role of α2‐adrenoceptors in adrenergic neurons, that is, α2‐autoreceptors, versus α2‐adrenoceptors in non‐adrenergic neurons, termed α2‐heteroreceptors. α2‐Autoreceptors are primarily required to limit release of noradrenaline from sympathetic nerves and adrenaline from adrenal chromaffin cells at rest. These receptors are desensitized upon chronic activation as it may for instance occur due to enhanced sympathetic activity during chronic heart failure. In contrast, pharmacological effects of acutely administered α2‐adrenoceptor agonist drugs essentially require α2‐heteroreceptors in non‐adrenergic neurons, including analgesia, sedation, hypothermia and anaesthetic‐sparing as well as bradycardia and hypotension. Thus a clear picture has emerged of the significance of auto‐ versus heteroreceptors in mediating the physiological functions of α2‐adrenoceptors and the pharmacological functions of α2‐adrenoceptor agonist drugs respectively.

Association of major depression with rare functional variants in norepinephrine transporter and serotonin1A receptor genes.

Dysregulations of central noradrenergic and serotonergic neurotransmission have been suggested to contribute to the pathogenesis of neuropsychiatric disorders such as depression. The norepinephrine transporter (NET; SLC6A2) and the serotonin (5‐HT)1A receptor (5‐HT1A receptor; HTR1A) play an important role in central nervous monoaminergic homeostasis. As shown previously, variations in the human NET and 5‐HT1A receptor genes can alter noradrenergic and serotonergic signaling in the brain: a single nucleotide polymorphism (SNP) in the coding region of the NET gene resulting in a F528C substitution increased plasma membrane expression of this NET variant, and a SNP in the human 5‐HT1A receptor gene leading to the R219L receptor variant almost abolished cellular signal transduction subsequent to receptor activation. The present study aimed at investigating whether these NET and 5‐HT1A receptor variants are associated with major depression (MD). The sample comprised 426 patients suffering from unipolar MD as well as 643 healthy control subjects for the variants of the 5‐HT1A receptor and the NET. Both SNPs were shown to be associated with MD. In conclusion, our results favor the hypothesis that monoaminergic neurotransmission in general and the F528C NET and R219L 5‐HT1A receptor variants in particular are involved in the pathogenesis of depression.

Presynaptic Metabotropic Receptors for Acetylcholine and Adrenaline/Noradrenaline

Presynaptic metabotropic receptors for acetylcholine and adrenaline/noradrenaline were first described more than three decades ago. Molecular cloning has resulted in the identification of five G protein-coupled muscarinic receptors (M(1) - M(5)) which mediate the biological effects of acetylcholine. Nine adrenoceptors (alpha(1ABD),alpha(2ABC),beta(123)) transmit adrenaline/noradrenaline signals between cells. The lack of sufficiently subtype-selective ligands has prevented identification of the physiological role and therapeutic potential of these receptor subtypes for a long time. Recently, mouse lines with targeted deletions for all muscarinic and adrenoceptor genes have been generated. This review summarizes the results from these gene-targeting studies with particular emphasis on presynaptic auto- and heteroreceptor functions of muscarinic and adrenergic receptors. Specific knowledge about the function of receptor subtypes will enhance our understanding of the physiological role of the cholinergic and adrenergic nervous system and open new avenues for subtype-selective therapeutic strategies.

Norepinephrine transporter knockout‐induced up‐regulation of brain alpha2A/C‐adrenergic receptors

The norepinephrine transporter (NET) is responsible for the rapid removal of norepinephrine released from sympathetic neurons; this release is controlled by inhibitory α2‐adrenergic receptors (α2ARs). Long‐term inhibition of the NET by antidepressants has been reported to change the density and function of pre‐ and postsynaptic ARs, which may contribute to the antidepressant effects of NET inhibitors such as desipramine. NET‐deficient (NET‐KO) mice have been described to behave like antidepressant‐treated mice. By means of quantitative real‐time PCR we show that mRNAs encoding the α2A‐adrenergic receptor (α2AAR) and the α2C‐adrenergic receptor (α2CAR) are up‐regulated in the brainstem, and that α2CAR mRNA is also elevated in the hippocampus and striatum of NET‐KO mice. These results were confirmed at the protein level by quantitative autoradiography. The NET‐KO mice showed enhanced binding of the selective α2AR antagonist [3H]RX821002 in several brain regions. Most robust increases (20–25%) in α2AR expression were observed in the hippocampus and in the striatum. Significant increases (16%) were also seen in the extended amygdala and thalamic structures. In an ‘in vivo’ test, the α2AR agonist clonidine (0.1 mg/kg) caused a significantly greater reduction of locomotor activity in NET‐KO mice than in wild‐type mice, showing the relevance of our findings at the functional level.