Lutz Hein

Novel Single Chain cAMP Sensors for Receptor-induced Signal Propagation

cAMP is a universal second messenger of many G-protein-coupled receptors and regulates a wide variety of cellular events. cAMP exerts its effects via cAMP-dependent protein kinase (PKA), cAMP-gated ion channels, and two isoforms of exchange protein directly activated by cAMP (Epac). Here we report the development of novel fluorescent indicators for cAMP based on the cAMP-binding domains of Epac and PKA. Fluorescence resonance energy transfer between variants of green fluorescent protein (enhanced cyan fluorescent protein and enhanced yellow fluorescent protein) fused directly to the cAMP-binding domains was used to analyze spatial and temporal aspects of cAMP-signaling in different cells. In contrast to previously developed PKA-based indicators, these probes are comprised of only a single binding site lacking cooperativity, catalytic properties, and interactions with other proteins and thereby allow us to easily image free intracellular cAMP and rapid signaling events. Rapid β-adrenergic receptor-induced cAMP signals were observed to travel with high speed (≈40 μm/s) throughout the entire cell body of hippocampal neurons and peritoneal macrophages. The developed indicators could be ubiquitously applied to studying cAMP, its physiological role and spatio-temporal regulation.

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.

Angiotensin-converting enzyme 2 protects from severe acute lung failure.

Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury, is a devastating clinical syndrome with a high mortality rate (30–60%) (refs 1–3). Predisposing factors for ARDS are diverse and include sepsis, aspiration, pneumonias and infections with the severe acute respiratory syndrome (SARS) coronavirus. At present, there are no effective drugs for improving the clinical outcome of ARDS. Angiotensin-converting enzyme (ACE) and ACE2 are homologues with different key functions in the renin–angiotensin system. ACE cleaves angiotensin I to generate angiotensin II, whereas ACE2 inactivates angiotensin II and is a negative regulator of the system. ACE2 has also recently been identified as a potential SARS virus receptor and is expressed in lungs. Here we report that ACE2 and the angiotensin II type 2 receptor (AT2) protect mice from severe acute lung injury induced by acid aspiration or sepsis. However, other components of the renin–angiotensin system, including ACE, angiotensin II and the angiotensin II type 1a receptor (AT1a), promote disease pathogenesis, induce lung oedemas and impair lung function. We show that mice deficient for Ace show markedly improved disease, and also that recombinant ACE2 can protect mice from severe acute lung injury. Our data identify a critical function for ACE2 in acute lung injury, pointing to a possible therapy for a syndrome affecting millions of people worldwide every year.

Direct evidence for a β1-adrenergic receptor–directed autoimmune attack as a cause of idiopathic dilated cardiomyopathy

Today, dilated cardiomyopathy (DCM) represents the main cause of severe heart failure and disability in younger adults and thus is a challenge for public health. About 30% of DCM cases are genetic in origin; however, the large majority of cases are sporadic, and a viral or immune pathogenesis is suspected. Following the established postulates for pathogenesis of autoimmune diseases, here we provide direct evidence that an autoimmune attack directed against the cardiac β1-adrenergic receptor may play a causal role in DCM. First, we immunized inbred rats against the second extracellular β1-receptor loop (β1-ECII; 100% sequence identity between human and rat) every month. All these rats developed first, receptor-stimulating anti–β1-ECII Ab’s and then, after 9 months, progressive severe left ventricular dilatation and dysfunction. Second, we transferred sera from anti–β1-ECII–positive and Ab-negative animals every month to healthy rats of the same strain. Strikingly, all anti–β1-ECII–transferred rats also developed a similar cardiomyopathic phenotype within a similar time frame, underlining the pathogenic potential of these receptor Ab’s. As a consequence, β1-adrenergic receptor–targeted autoimmune DCM should now be categorized with other known receptor Ab-mediated autoimmune diseases, such as Graves disease or myasthenia gravis. Although carried out in an experimental animal model, our findings should further encourage the development of therapeutic strategies that combat harmful anti–β1-ECII in receptor Ab–positive DCM patients.

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.

Inhibition of Na+-H+ Exchange Prevents Hypertrophy, Fibrosis, and Heart Failure in β1-Adrenergic Receptor Transgenic Mice

Chronic stimulation of the &bgr;1-adrenergic receptor leads to hypertrophy and heart failure in &bgr;1-adrenergic receptor transgenic mice and contributes to disease progression in heart failure patients. The cellular mechanisms underlying these detrimental effects are largely unknown. In this study, we have identified the cardiac Na+-H+ exchanger (NHE1) as a novel mediator of adrenergically induced heart failure. &bgr;1-Adrenergic receptor transgenic mice showed upregulation of both NHE1 mRNA (+140±6%) and protein (+42±19%). In order to test whether increased NHE1 is causally related to &bgr;1-adrenergic–induced hypertrophy, fibrosis, and heart failure, &bgr;1-adrenergic receptor transgenic (TG) and wild-type (WT) littermates were treated with a diet containing 6000 ppm of the NHE1 inhibitor cariporide or control chow for 8 months. There was significant hypertrophy of cardiac myocytes in &bgr;1-adrenergic receptor transgenic mice (2.3-fold increase in myocyte cross-sectional area), which was virtually absent in cariporide-fed animals. Interstitial fibrosis was prominent throughout the left ventricular wall in nontreated &bgr;1-adrenergic receptor transgenic mice (4.8-fold increase in collagen volume fraction); cariporide treatment completely prevented this development of fibrosis. Left ventricular catheterization showed that cariporide also prevented the loss of contractile function in &bgr;1-adrenergic receptor transgenic mice: whereas untreated transgenic mice showed a significant decrease in left ventricular contractility (5250±570 mm Hg/s TG versus 7360±540 mm Hg/s WT, dp/dtmax), this decrease was completely prevented by cariporide (8150±520 mm Hg/s TG cariporide). Inhibition of NHE1 prevented the development of heart failure in &bgr;1-receptor transgenic mice. We conclude that the cardiac Na+-H+ exchanger 1 is essential for the detrimental cardiac effects of chronic &bgr;1-receptor stimulation in the heart.

Dobutamine-Stress Magnetic Resonance Microimaging in Mice Acute Changes of Cardiac Geometry and Function in Normal and Failing Murine Hearts

Abstract— The aim of this study was to assess the capability of MRI to characterize systolic and diastolic function in normal and chronically failing mouse hearts in vivo at rest and during inotropic stimulation. Applying an ECG-gated FLASH-cine sequence, MRI at 7 T was performed at rest and after administration of 1.5 &mgr;g/g IP dobutamine. There was a significant increase of heart rate, cardiac output, and ejection fraction and significant decrease of end-diastolic and end-systolic left ventricular (LV) volumes (P <0.01 each) in normal mice during inotropic stimulation. In mice with heart failure due to chronic myocardial infarction (MI), MRI at rest revealed gross LV dilatation. There was a significant decrease of LV ejection fraction in infarcted mice (29%) versus sham mice (58%). Mice with MI showed a significantly reduced maximum LV ejection rate (P <0.001) and LV filling rate (P <0.01) and no increase of LV dynamics during dobutamine action, indicating loss of contractile and relaxation reserve. In 4-month-old transgenic mice with cardiospecific overexpression of the &bgr;1-adrenergic receptor, which at this early stage do not show abnormalities of resting cardiac function, LV filling rate failed to increase after dobutamine stress (transgenic, 0.19±0.03 &mgr;L/ms; wild type, 0.36±0.01 &mgr;L/ms;P <0.01). Thus, MRI unmasked diastolic dysfunction during dobutamine stress. Dobutamine-stress MRI allows noninvasive assessment of systolic and diastolic components of heart failure. This study shows that MRI can demonstrate loss of inotropic and lusitropic response in mice with MI and can unmask diastolic dysfunction as an early sign of cardiac dysfunction in a transgenic mouse model of heart failure.

Adrenoceptors and signal transduction in neurons

The adrenergic system is an essential regulator of neuronal, endocrine, cardiovascular, vegetative, and metabolic functions. The endogenous catecholamines epinephrine and norepinephrine activate G-protein-coupled receptors to transmit their signal across the plasma membrane. These adrenoceptors can be divided into three different groups: the α1-receptors (α1A, α1B, α1D), α2-receptors (α2A, α2B, α2C), and β-receptors (β1, β2, β3). This review summarizes recent findings in the field of adrenoceptor signaling in neurons and includes a discussion of receptor-associated proteins, receptor dimerization, subcellular trafficking, and fluorescence optical methods for studying the kinetics of adrenergic signaling. Spatio-temporal imaging may become an important future tool for identifying the physiological significance of these complex signaling mechanisms in vivo. Gene-targeted mouse models carrying deletions in α2-adrenoceptor have provided detailed insights into specific neuronal functions of the three α2-receptor subtypes.

Deletion of Cardiomyocyte Mineralocorticoid Receptor Ameliorates Adverse Remodeling After Myocardial Infarction

Background— Mineralocorticoid receptor (MR) blockade improves morbidity and mortality among patients with heart failure; however, the underlying mechanisms are still under investigation. We studied left ventricular remodeling after myocardial infarction in mice with cardiomyocyte-specific inactivation of the MR gene (MRMLCCre) that were generated with a conditional MR allele (MRflox) in combination with a transgene expressing Cre recombinase under control of the myosin light-chain (MLC2a) gene promoter. Methods and Results— Control (MRflox/flox, MRflox/wt) and MRMLCCre mice underwent coronary artery ligation. MR ablation had no detectable baseline effect on cardiac morphology and function. The progressive left ventricular chamber enlargement and functional deterioration in infarcted control mice, detected by echocardiography and conductance catheter analysis during the 8-week observation period, were substantially attenuated in MRMLCCre mice. Chronically infarcted MRMLCCre mice displayed attenuated pulmonary edema, reduced cardiac hypertrophy, increased capillary density, and reduced accumulation of extracellular matrix proteins in the surviving left ventricular myocardium. Moreover, cardiomyocyte-specific MR ablation prevented the increases in myocardial and mitochondrial O2·− production and upregulation of the NADPH oxidase subunits Nox2 and Nox4. At 7 days, MRMLCCre mice exhibited enhanced infarct neovessel formation and collagen structural organization associated with reduced infarct expansion. Mechanistically, cardiomyocytes lacking MR displayed accelerated stress-induced activation and subsequent suppression of nuclear factor-&kgr;B and reduced apoptosis early after myocardial infarction. Conclusion— Cardiomyocyte-specific MR deficiency improved infarct healing and prevented progressive adverse cardiac remodeling, contractile dysfunction, and molecular alterations in ischemic heart failure, highlighting the importance of cardiomyocyte MR for heart failure development and progression.

Two alpha(2)-adrenergic receptor subtypes, alpha(2A) and alpha(2C), inhibit transmitter release in the brain of gene-targeted mice.

Abstract α 2 -Adrenergic receptors play an essential role in regulating neurotransmitter release from sympathetic nerves and from adrenergic neurons in the CNS. However, the role of each of the three highly homologous α 2 -adrenergic receptor subtypes (α 2A , α 2B , α 2C ) in this process has not been determined unequivocally. To address this question, the regulation of norepinephrine and dopamine release was studied in mice carrying deletions in the genes encoding the three α 2 -adrenergic receptor subtypes. Autoradiography and radioligand binding studies showed that α 2 -receptor density in α 2A -deficient brains was decreased to 9±1% of the respective wild-type value, whereas α 2 -receptor levels were reduced to 83±4% in α 2C -deficient mice. These results indicate that approximately 90% of mouse brain α 2 -receptors belong to the α 2A subtype and 10% are α 2C -receptors. In isolated brain cortex slices from wild-type mice a non-subtype-selective α 2 -receptor agonist inhibited release of [ 3 H]norepinephrine by maximally 96%. Similarly, release of [ 3 H]dopamine from isolated basal ganglion slices was inhibited by 76% by an α 2 -receptor agonist. In α 2A -receptor-deficient mice, the inhibitory effect of the α 2 -receptor agonist on norepinephrine and dopamine release was significantly reduced but not abolished. Only in tissues from mice lacking both α 2A - and α 2C -receptors was no α 2 -receptor agonist effect on transmitter release observed. The time course of onset of presynaptic inhibition of norepinephrine release was much faster for the α 2A -receptor than for the α 2C -subtype. After prolonged stimulation with norepinephrine, presynaptic α 2C -adrenergic receptors were desensitized. From these data we suggest that two functionally distinct α 2 -adrenergic receptor subtypes, α 2A and α 2C , operate as presynaptic inhibitory receptors regulating neurotransmitter release in the mouse CNS.