Nadine Beetz

Acquisition of the contractile phenotype by murine arterial smooth muscle cells depends on the Mir143/145 gene cluster

VSMCs respond to changes in the local environment by adjusting their phenotype from contractile to synthetic, a phenomenon known as phenotypic modulation or switching. Failure of VSMCs to acquire and maintain the contractile phenotype plays a key role in a number of major human diseases, including arteriosclerosis. Although several regulatory circuits that control differentiation of SMCs have been identified, the decisive mechanisms that govern phenotypic modulation remain unknown. Here, we demonstrate that the mouse miR-143/145 cluster, expression of which is confined to SMCs during development, is required for VSMC acquisition of the contractile phenotype. VSMCs from miR-143/145-deficient mice were locked in the synthetic state, which incapacitated their contractile abilities and favored neointimal lesion development. Unbiased high-throughput, quantitative, mass spectrometry-based proteomics using reference mice labeled with stable isotopes allowed identification of miR-143/145 targets; these included angiotensin-converting enzyme (ACE), which might affect both the synthetic phenotype and contractile functions of VSMCs. Pharmacological inhibition of either ACE or the AT1 receptor partially reversed vascular dysfunction and normalized gene expression in miR-143/145-deficient mice. We conclude that manipulation of miR-143/145 expression may offer a new approach for influencing vascular repair and attenuating arteriosclerotic pathogenesis.

Impaired Heart Contractility in Apelin Gene–Deficient Mice Associated With Aging and Pressure Overload

Apelin constitutes a novel endogenous peptide system suggested to be involved in a broad range of physiological functions, including cardiovascular function, heart development, control of fluid homeostasis, and obesity. Apelin is also a catalytic substrate for angiotensin-converting enzyme 2, the key severe acute respiratory syndrome receptor. The in vivo physiological role of Apelin is still elusive. Here we report the generation of Apelin gene–targeted mice. Apelin mutant mice are viable and fertile, appear healthy, and exhibit normal body weight, water and food intake, heart rates, and heart morphology. Intriguingly, aged Apelin knockout mice developed progressive impairment of cardiac contractility associated with systolic dysfunction in the absence of histological abnormalities. We also report that pressure overload induces upregulation of Apelin expression in the heart. Importantly, in pressure overload–induced heart failure, loss of Apelin did not significantly affect the hypertrophy response, but Apelin mutant mice developed progressive heart failure. Global gene expression arrays and hierarchical clustering of differentially expressed genes in hearts of banded Apelin−/y and Apelin+/y mice showed concerted upregulation of genes involved in extracellular matrix remodeling and muscle contraction. These genetic data show that the endogenous peptide Apelin is crucial to maintain cardiac contractility in pressure overload and aging.

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.

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.

Phosducin influences sympathetic activity and prevents stress-induced hypertension in humans and mice

Hypertension and its complications represent leading causes of morbidity and mortality. Although the cause of hypertension is unknown in most patients, genetic factors are recognized as contributing significantly to an individuals lifetime risk of developing the condition. Here, we investigated the role of the G protein regulator phosducin (Pdc) in hypertension. Mice with a targeted deletion of the gene encoding Pdc (Pdc-/- mice) had increased blood pressure despite normal cardiac function and vascular reactivity, and displayed elevated catecholamine turnover in the peripheral sympathetic system. Isolated postganglionic sympathetic neurons from Pdc-/- mice showed prolonged action potential firing after stimulation with acetylcholine and increased firing frequencies during membrane depolarization. Furthermore, Pdc-/- mice displayed exaggerated increases in blood pressure in response to post-operative stress. Candidate gene-based association studies in 2 different human populations revealed several SNPs in the PDC gene to be associated with stress-dependent blood pressure phenotypes. Individuals homozygous for the G allele of an intronic PDC SNP (rs12402521) had 12-15 mmHg higher blood pressure than those carrying the A allele. These findings demonstrate that PDC is an important modulator of sympathetic activity and blood pressure and may thus represent a promising target for treatment of stress-dependent hypertension.

Direct Inhibition of Cardiac Hyperpolarization-Activated Cyclic Nucleotide–Gated Pacemaker Channels by Clonidine

Background— Inhibition of cardiac sympathetic tone represents an important strategy for treatment of cardiovascular disease, including arrhythmia, coronary heart disease, and chronic heart failure. Activation of presynaptic α2-adrenoceptors is the most widely accepted mechanism of action of the antisympathetic drug clonidine; however, other target proteins have been postulated to contribute to the in vivo actions of clonidine. Methods and Results— To test whether clonidine elicits pharmacological effects independent of α2-adrenoceptors, we have generated mice with a targeted deletion of all 3 α2-adrenoceptor subtypes (α2ABC−/−). α2ABC−/− mice were completely unresponsive to the analgesic and hypnotic effects of clonidine; however, clonidine significantly lowered heart rate in α2ABC−/− mice by up to 150 bpm. Clonidine-induced bradycardia in conscious α2ABC−/− mice was 32.3% (10 &mgr;g/kg) and 26.6% (100 &mgr;g/kg) of the effect in wild-type mice. A similar bradycardic effect of clonidine was observed in isolated spontaneously beating right atria from α2ABC-knockout and wild-type mice. Clonidine inhibited the native pacemaker current (If) in isolated sinoatrial node pacemaker cells and the If-generating hyperpolarization-activated cyclic nucleotide–gated (HCN) 2 and HCN4 channels in transfected HEK293 cells. As a consequence of blocking If, clonidine reduced the slope of the diastolic depolarization and the frequency of pacemaker potentials in sinoatrial node cells from wild-type and α2ABC-knockout mice. Conclusions— Direct inhibition of cardiac HCN pacemaker channels contributes to the bradycardic effects of clonidine gene-targeted mice in vivo, and thus, clonidine-like drugs represent novel structures for future HCN channel inhibitors.

Adrenergic Repression of the Epigenetic Reader MeCP2 Facilitates Cardiac Adaptation in Chronic Heart Failure.

Supplemental Digital Content is available in the text.

Transgenic simulation of human heart failure-like L-type Ca2+-channels: implications for fibrosis and heart rate in mice

AIMS Cardiac L-type Ca(2+)-currents show distinct alterations in chronic heart failure, including increased single-channel activity and blunted adrenergic stimulation, but minor changes of whole-cell currents. Expression of L-type Ca(2+)-channel beta(2)-subunits is enhanced in human failing hearts. In order to determine whether prolonged alteration of Ca(2+)-channel gating by beta(2)-subunits contributes to heart failure pathogenesis, we generated and characterized transgenic mice with cardiac overexpression of a beta(2a)-subunit or the pore Ca(v)1.2 or both, respectively. METHODS AND RESULTS Four weeks induction of cardiac-specific overexpression of rat beta(2a)-subunits shifted steady-state activation and inactivation of whole-cell currents towards more negative potentials, leading to increased Ca(2+)-current density at more negative test potentials. Activity of single Ca(2+)-channels was increased in myocytes isolated from beta(2a)-transgenic mice. Ca(2+)-current stimulation by 8-Br-cAMP and okadaic acid was blunted in beta(2a)-transgenic myocytes. In vivo investigation revealed hypotension and bradycardia upon Ca(v)1.2-transgene expression but not in mice only overexpressing beta(2a). Double-transgenics showed cardiac arrhythmia. Interstitial fibrosis was aggravated by the beta(2a)-transgene compared with Ca(v)1.2-transgene expression alone. Overt cardiac hypertrophy was not observed in any model. CONCLUSION Cardiac overexpression of a Ca(2+)-channel beta(2a)-subunit alone is sufficient to induce Ca(2+)-channel properties characteristic of chronic human heart failure. beta(2a)-overexpression by itself did not induce cardiac hypertrophy or contractile dysfunction, but aggravated the development of arrhythmia and fibrosis in Ca(v)1.2-transgenic mice.

Modulation of α2-adrenoceptor functions by heterotrimeric Gαi protein isoforms

Subtype diversity of heterotrimeric G proteins and G protein-coupled receptors enables a wide spectrum of signal transduction. However, the significance of isoforms within receptor or G protein subfamilies has not been fully elucidated. In the present study, we have tested whether α2-adrenoceptors require specific Gα isoforms for their function in vivo. In particular, we analyzed the role of the highly homologous Gαi isoforms, Gαi1, Gαi2, and Gαi3, in typical α2-adrenoceptor-controlled functions. Mice with targeted deletions in the genes encoding Gαi1, Gαi2, or Gαi3 were used to test the effects of α2-adrenoceptor stimulation by the agonist medetomidine. The α2-adrenoceptor agonist medetomidine inhibited [3H]norepinephrine release from isolated prefrontal brain cortex or cardiac atria tissue specimens with similar potency and efficacy in tissues from wild-type or Gαi-deficient mice. In vivo, bradycardia, hypotension, induction of sleep, antinociception, and hypothermia induced by α2-adrenoceptor activation did not differ between wild-type and Gαi-knockout mice. However, the effects of the α2-agonists medetomidine or 5-bromo-6-(2-imidazolin-2-ylamino)quin-oxaline tartrate (UK14,304) on spontaneous locomotor activity or anesthetic sparing were reduced or absent, respectively, in mice lacking Gαi2. In microdissected locus coeruleus neurons or postganglionic sympathetic neurons from stellate ganglia, all three Gαi subunits were expressed as determined by quantitative reverse transcription-polymerase chain reaction, with Gαi1 and Gαi2 dominating over Gαi3. Functional redundancy of the highly homologous Gαi isoforms may predominate over specificity to regulate distinct intracellular pathways downstream of α2-adrenoceptors in vivo. In contrast, inhibition of locomotor activity and anesthetic sparing may be elicited by a specific coupling of α2A-adrenoceptors via the Gαi2 isoform to intracellular pathways.

Modulation of alpha2-adrenoceptor functions by heterotrimeric Galphai protein isoforms.

Subtype diversity of heterotrimeric G proteins and G protein-coupled receptors enables a wide spectrum of signal transduction. However, the significance of isoforms within receptor or G protein subfamilies has not been fully elucidated. In the present study, we have tested whether α2-adrenoceptors require specific Gα isoforms for their function in vivo. In particular, we analyzed the role of the highly homologous Gαi isoforms, Gαi1, Gαi2, and Gαi3, in typical α2-adrenoceptor-controlled functions. Mice with targeted deletions in the genes encoding Gαi1, Gαi2, or Gαi3 were used to test the effects of α2-adrenoceptor stimulation by the agonist medetomidine. The α2-adrenoceptor agonist medetomidine inhibited [3H]norepinephrine release from isolated prefrontal brain cortex or cardiac atria tissue specimens with similar potency and efficacy in tissues from wild-type or Gαi-deficient mice. In vivo, bradycardia, hypotension, induction of sleep, antinociception, and hypothermia induced by α2-adrenoceptor activation did not differ between wild-type and Gαi-knockout mice. However, the effects of the α2-agonists medetomidine or 5-bromo-6-(2-imidazolin-2-ylamino)quin-oxaline tartrate (UK14,304) on spontaneous locomotor activity or anesthetic sparing were reduced or absent, respectively, in mice lacking Gαi2. In microdissected locus coeruleus neurons or postganglionic sympathetic neurons from stellate ganglia, all three Gαi subunits were expressed as determined by quantitative reverse transcription-polymerase chain reaction, with Gαi1 and Gαi2 dominating over Gαi3. Functional redundancy of the highly homologous Gαi isoforms may predominate over specificity to regulate distinct intracellular pathways downstream of α2-adrenoceptors in vivo. In contrast, inhibition of locomotor activity and anesthetic sparing may be elicited by a specific coupling of α2A-adrenoceptors via the Gαi2 isoform to intracellular pathways.