Anne-Ulrike Trendelenburg

GDF11 Increases with Age and Inhibits Skeletal Muscle Regeneration.

Age-related frailty may be due to decreased skeletal muscle regeneration. The role of TGF-β molecules myostatin and GDF11 in regeneration is unclear. Recent studies showed an age-related decrease in GDF11 and that GDF11 treatment improves muscle regeneration, which were contrary to prior studies. We now show that these recent claims are not reproducible and the reagents previously used to detect GDF11 are not GDF11 specific. We develop a GDF11-specific immunoassay and show a trend toward increased GDF11 levels in sera of aged rats and humans. GDF11 mRNA increases in rat muscle with age. Mechanistically, GDF11 and myostatin both induce SMAD2/3 phosphorylation, inhibit myoblast differentiation, and regulate identical downstream signaling. GDF11 significantly inhibited muscle regeneration and decreased satellite cell expansion in mice. Given early data in humans showing a trend for an age-related increase, GDF11 could be a target for pharmacologic blockade to treat age-related sarcopenia.

Treatment of sporadic inclusion body myositis with bimagrumab

Objective: To study activin signaling and its blockade in sporadic inclusion body myositis (sIBM) through translational studies and a randomized controlled trial. Methods: We measured transforming growth factor β signaling by SMAD2/3 phosphorylation in muscle biopsies of 50 patients with neuromuscular disease (17 with sIBM). We tested inhibition of activin receptors IIA and IIB (ActRII) in 14 patients with sIBM using one dose of bimagrumab (n = 11) or placebo (n = 3). The primary outcome was the change in right thigh muscle volume by MRI at 8 weeks. Lean body mass, strength, and function were secondary outcomes. Twelve of the patients (10 bimagrumab, 2 placebo) participated in a subsequent 16-week observation phase. Results: Muscle SMAD2/3 phosphorylation was higher in sIBM than in other muscle diseases studied (p = 0.003). Eight weeks after dosing, the bimagrumab-treated patients increased thigh muscle volume (right leg +6.5% compared with placebo, p = 0.024; left leg +7.6%, p = 0.009) and lean body mass (+5.7% compared with placebo, p = 0.014). Subsequently, bimagrumab-treated patients had improved 6-minute walking distance, which peaked at 16 weeks (+14.6%, p = 0.008) compared with placebo. There were no serious adverse events; the main adverse events with bimagrumab were mild acne and transient involuntary muscle contractions. Conclusions: Transforming growth factor β superfamily signaling, at least through ActRII, is implicated in the pathophysiology of sIBM. Inhibition of ActRII increased muscle mass and function in this pilot trial, offering a potential novel treatment of sIBM. Classification of evidence: This study provides Class I evidence that for patients with inclusion body myositis, bimagrumab increases thigh muscle volume at 8 weeks.

Presynaptic α2-autoreceptors in brain cortex: α2D in the rat and α2A in the rabbit

SummaryPresynaptic α2-autoreceptors in rat and rabbit brain cortex were compared by means of antagonists and agonists. Brain cortex slices were preincubated with [3H]-noradrenaline and then superfused and stimulated by 3 (rat) or 4 (rabbit) pulses at a frequency of 100 Hz.The α2-adrenoceptor agonist bromoxidine (UK 14 304) reduced the electrically evoked overflow of tritium with EC50 values of 4.5 nmol/l in the rat and 0.7 nmol/l in the rabbit. The antagonists phentolamine, 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole (BRL 44408), rauwolscine, 1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo(c,f)imidazo(1,5-a)azepine (BRL 41992), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4, 5-tetrahydro-3-benzazepine (SKF 104078), imiloxan, prazosin and corynanthine did not per se increase the evoked overflow of tritium but shifted the concentration-inhibition curve of bromoxidine to the right in a manner compatible with competitive antagonism. Up to 4 concentrations of each antagonist were used to determine its dissociation constant KD. The KD values correlated only weakly between the rat and the rabbit. Dissociation constants KA of bromoxidine were calculated from equieffective concentrations in unpretreated brain slices and slices in which part of the α2-adrenoceptors had been irreversibly blocked by phenoxybenzamine. The KA value was 123 nmol/l in the rat and 7.2 nmol/l in the rabbit.The results confirm the species difference between rat and rabbit brain presynaptic α2-autoreceptors. Comparison with data from the literature indicates that the rat brain autoreceptors can be equated with the α2D subtype as defined by radioligand binding, whereas the rabbit brain autoreceptors conform to the α2A subtype. For example, the antagonist affinities for the rat autoreceptors correlate with their binding affinities for the gene product of α2-RG20, the putative rat α2D-adrenoceptor gene (r = 0.97; P<0.01), but not with their binding affinities for the gene product of α2-C10, the putative human α2A-adrenoceptor gene. Conversely, the rabbit autoreceptors correlate with the α2-C10 (r = 0.98; P<0.001) but not with the α2-RG20 gene product. Since presynaptic α2-autoreceptors are also α2D in rat submaxillary gland and perhaps vas deferens and α2A in rabbit pulmonary artery, the possibility arises that the majority of α2-autoreceptors generally are α2D in the rat and α2A in the rabbit. Moreover, receptors of the α2A/D group generally may be the main mammalian α2-autoreceptors.

PRESYNAPTIC ALPHA 2A-ADRENOCEPTORS INHIBIT THE RELEASE OF ENDOGENOUS DOPAMINE IN RABBIT CAUDATE NUCLEUS SLICES

Abstractα2-Adrenoceptors modulating the release of dopamine were identified and characterized in slices of the head of the rabbit caudate nucleus. Release of endogenous dopamine was measured by fast cyclic voltammetry as the increase in the extracellular concentration of dopamine elicited by electrical stimulation. The electrochemical signal was identified as dopamine by means of the oxidation potential, the voltammogram and the fact that the signal was not changed by desipramine, which inhibits the high affinity uptake of noradrenaline, but was greatly increased by nomifensine, which in addition inhibits the high affinity uptake of dopamine.Stimulation by 6 pulses/100 Hz increased the extracellular concentration of dopamine by about 85 nM. The selective α2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced this release with an EC50 of 173 nM and by maximally 75%. The α2-adrenoceptor agonists clonidine and oxymetazoline only tended to cause a decrease. Six drugs, including oxymetazoline, were tested as antagonists against UK 14,304. Their order of antagonist potency (pKD values in brackets) was rauwolscine (8.0) > oxymetazoline (7.5) > 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101; 7.3) > phentolamine (7.1) > corynanthine (5.1) ≈ prazosin (< 6). Given alone, the antagonists did not change the release of dopamine elicited by 6 pulses/100 Hz, and the same was true for the dopamine receptor antagonist sulpiride. When caudate slices were stimulated by 10 pulses/1 Hz, sulpiride increased the release of dopamine. Desipramine and rauwolscine, in contrast, again caused no change.It is concluded that dopaminergic axons in the rabbit caudate nucleus possess release-inhibiting α2-adrenoceptors. The antagonist affinities indicate that they belong to the α2A subtype. In this, they agree with all presynaptic α2-autoreceptors studied so far in rabbits as well as with the α2-heteroreceptors modulating the release of serotonin in rabbit brain cortex, suggesting that at least the majority of presynaptic α2-adrenoceptors in the rabbit are α2A. The agonist sensitivity of the caudate presynaptic α2-adrenoceptors is low in comparison with cerebrocortical presynaptic α2-autoreceptors, possibly due to absence of a receptor reserve.

A study of presynaptic alpha2-autoreceptors in alpha2A/D-, alpha2B- and alpha2C-adrenoceptor-deficient mice.

Abstract. The function of presynaptic α2-autoreceptors was studied in the hippocampus, occipito-parietal cortex, atria and vas deferens of NMRI mice, mice in which the α2A/D-, the α2B- or α2C-adrenoceptor gene had been disrupted (α2A/DKO, α2BKO and α2CKO, respectively), and the wildtype mice from which the knockout animals had been generated. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically.The α2-adrenoceptor agonist medetomidine reduced the electrically evoked overflow of tritium in all tissues from all mouse strains (stimulation with single pulses or single high-frequency pulse trains, called POPs, i.e. pulse patterns leading to minimal autoinhibition). The effects of medetomidine did not differ in NMRI, wildtype, α2BKO and α2CKO mice but were greatly reduced in α2A/DKO brain preparations and to a lesser extent in α2A/DKO atria and vasa deferentia. Six drugs were tested as antagonists against medetomidine. Their pKd values indicated that the hippocampal and occipito-parietal α2-autoreceptors in NMRI and wildtype mice were α2D (the rodent variant of the α2A/D-adrenoceptor) whereas the atrial and vas deferens α2-autoreceptors in NMRI and wildtype mice could not be identified with a single α2 subtype. Deletion of the α2A/D gene changed the pKd values in all tissues so that they now reflected α2C properties, whereas deletion of the α2C gene changed the pKd values in atria and vasa deferentia so that they now had α2D properties (as they had in NMRI and wildtype brain preparations). Autoinhibition by released noradrenaline was created using trains of up to 64 pulses or up to 4 POPs, and the overflow-enhancing effect of the α2 antagonist rauwolscine was determined. Results did not differ, irrespective of whether preparations were obtained from NMRI, wildtype, α2BKO or α2CKO mice: the overflow of tritium elicited by p pulses or POPs was much smaller than p times the overflow elicited by a single pulse or POP, and rauwolscine greatly increased the evoked overflow. Results differed, however, in tissues taken from α2A/DKO mice: in these tissues, the overflow of tritium elicited by p pulses or POPs was close to p times the overflow elicited by a single pulse or POP, and rauwolscine did not increase the evoked overflow of tritiumor increased it only marginally. When a greater degree of autoinhibition was produced in atria and vasa deferentia by stimulation with 120 pulses, both disruption of the α2A/D gene and disruption of the α2C gene but not disruption of the α2B gene attenuated the overflow-enhancing effects of phentolamine and rauwolscine. In NMRI and wildtype atria and vasa deferentia, the relative potencies of phentolamine and rauwolscine at enhancing the evoked overflow were not easily compatible with a single α2 subtype. In α2A/DKO atria and vasa deferentia, the relative potencies of phentolamine and rauwolscine indicated that the autoinhibition-mediating receptors were α2C, whereas in α2CKO atria and vasa deferentia the relative potencies indicated that the autoinhibition-mediating receptors were α2D.It is concluded that α2-autoreceptors function identically in NMRI mice and the wildtype mice from which the receptor-deficient animals had been generated. There is no evidence from the experiments for any contribution of α2B-adrenoceptors to autoreceptor function. The main presynaptic α2-autoreceptors are α2A/D, both as sites of action of exogenous agonists and as sites of action of previously released noradrenaline. However, there are in addition non-α2A/D-, probably α2C-autoreceptors. They are less prominent in mediating the inhibitory effects of exogenous agonists and the negative feedback effect of released noradrenaline. They operate not only after deletion of the α2A/D-adrenoceptors but also in normal (NMRI, wildtype) mice without gene deletion.

Blockade of the Activin Receptor IIB Activates Functional Brown Adipogenesis and Thermogenesis by Inducing Mitochondrial Oxidative Metabolism

ABSTRACT Brown adipose tissue (BAT) is a key tissue for energy expenditure via fat and glucose oxidation for thermogenesis. In this study, we demonstrate that the myostatin/activin receptor IIB (ActRIIB) pathway, which serves as an important negative regulator of muscle growth, is also a negative regulator of brown adipocyte differentiation. In parallel to the anticipated hypertrophy of skeletal muscle, the pharmacological inhibition of ActRIIB in mice, using a neutralizing antibody, increases the amount of BAT without directly affecting white adipose tissue. Mechanistically, inhibition of ActRIIB inhibits Smad3 signaling and activates the expression of myoglobin and PGC-1 coregulators in brown adipocytes. Consequently, ActRIIB blockade in brown adipose tissue enhances mitochondrial function and uncoupled respiration, translating into beneficial functional consequences, including enhanced cold tolerance and increased energy expenditure. Importantly, ActRIIB inhibition enhanced energy expenditure only at ambient temperature or in the cold and not at thermoneutrality, where nonshivering thermogenesis is minimal, strongly suggesting that brown fat activation plays a prominent role in the metabolic actions of ActRIIB inhibition.

All three α2-adrenoceptor types serve as autoreceptors in postganglionic sympathetic neurons

Postganglionic sympathetic neurons and brain noradrenergic neurons use α2A- and α2C-adrenoceptors as presynaptic autoreceptors. The present experiments were carried out in order to see whether they possess presynaptic α2B-autoreceptors as well. Pieces of atria, vasa deferentia, the occipito-parietal cortex and the hippocampus were prepared from either wildtype (WT) mice or mice in which both the α2A- and the α2C-adrenoceptor gene had been disrupted (α2ACKO). The pieces were incubated with 3H-noradrenaline and then superfused and stimulated electrically. In a first series of experiments, single pulses or brief, autoinhibition-poor pulse trains were used for stimulation. The α2-adrenoceptor agonist UK 14,304 (brimonidine) reduced the evoked overflow of tritium in all four tissues from WT mice but did not change it in any tissue from α2ACKO mice. A different pattern was obtained with medetomidine as α2 agonist. Like UK 14,304, medetomidine reduced the evoked overflow of tritium in all four tissues from WT mice and did not affect overflow in brain slices from α2ACKO mice; however, in contrast to UK 14,304, medetomidine reduced evoked overflow also in atrial and vas deferens pieces from α2ACKO mice, although with a lower maximum and potency than in WT preparations. The α-adrenoceptor antagonists rauwolscine, phentolamine, prazosin, spiroxatrine and WB 4101 shifted the concentration-response curve of medetomidine in α2ACKO atria and vasa deferentia to the right. The pKd values of the five antagonists against medetomidine in α2ACKO atria and vasa deferentia correlated with pKd values at prototypical α2B radioligand binding sites but not at α2A or α2C binding sites. In a second series of experiments, autoinhibition-rich pulse trains were used for stimulation. Under these conditions, rauwolscine and phentolamine increased the evoked overflow of tritium from α2ACKO atrial and vas deferens pieces but not from α2ACKO brain slices. The increase was smaller (by 40% in atria and by 70% in the vas deferens) than previously observed in WT preparations (by 200–400%). In a last series of experiments, mRNA for the α2B-adrenoceptor was demonstrated by RT-PCR in thoracolumbar sympathetic ganglia from WT, α2AKO, α2CKO and α2ACKO mice but not from α2BKO mice. The results show that brain noradrenergic neurons express only α2A- and α2C-adrenoceptors as autoreceptors. Postganglionic sympathetic neurons, however, can express α2B-adrenoceptors as presynaptic autoreceptors as well. The α2B-autoreceptors are activated by medetomidine but not by UK 14,304. They are also activated by previously released noradrenaline. The two-α2-autoreceptor hypothesis has to be replaced by a three-autoreceptor hypothesis for postganglionic sympathetic neurons.

α2-Adrenoceptors modulating neuronal serotonin release: a study in α2-adrenoceptor subtype-deficient mice

The release‐inhibiting α2‐adrenoceptors of cerebral serotoninergic axons were studied in mice. Slices of the hippocampus or the occipito‐parietal cortex from NMRI mice, from mice lacking the α2A/D‐, the α2B‐, the α2C‐ or both the α2A/D‐ and the α2C‐adrenoceptor, and from mice sharing the genetic background of the receptor‐deficient animals (WT) were preincubated with [3H]‐serotonin and then superfused and stimulated electrically, in most experiments by trains of 8 pulses at 100 Hz. The concentration‐response curves of the α2‐adrenoceptor agonist medetomidine were virtually identical in hippocampal slices from NMRI and WT mice, with maximally 70% inhibition and an EC50 of about 2 nM. In hippocampal slices from NMRI mice, phentolamine and rauwolscine were equipotent antagonists against medetomidine. The effect of medetomidine was greatly reduced, with maximally 20% inhibition, in hippocampal slices from α2A/D‐adrenoceptor‐deficient mice; was slightly reduced, with maximally 59% inhibition, in hippocampal slices from α2C‐adrenoceptor‐deficient mice; was not changed in hippocampal slices from α2B‐adrenoceptor‐deficient mice; and was abolished in hippocampal slices from mice lacking both the α2A/D‐ and the α2C‐adrenoceptor. Similar results were obtained in: (i) occipito‐parietal slices from NMRI and α2A/D‐adrenoceptor‐deficient mice and (ii) hippocampal slices that were preincubated with [3H]‐serotonin in the presence of oxaprotiline to rule out cross‐labelling of noradrenergic axons. The serotoninergic axons of the mouse brain possess both α2A/D‐heteroreceptors, which predominate, and α2C‐heteroreceptors but lack α2B‐adrenoceptors. The situation resembles the coexistence of α2A/D‐ and α2C‐autoreceptors but lack of α2B‐autoreceptors at the noradrenergic axons of mice.

Heterogeneity of presynaptic muscarinic receptors mediating inhibition of sympathetic transmitter release: a study with M2- and M4-receptor-deficient mice

Presynaptic muscarinic receptors modulate sympathetic transmitter release. The goal of the present study was to identify the muscarinic receptor subtype(s) mediating inhibition of sympathetic transmitter release in mouse atria, urinary bladder and vas deferens. To address this question, electrically evoked noradrenaline release was assessed using tissue preparations from NMRI, M2‐ and M4‐knockout, and the corresponding M2‐ and M4‐wildtype mice, after preincubation with 3H‐noradrenaline. The muscarinic agonist carbachol decreased evoked tritium overflow (20 pulses/50 Hz) in each tissue and strain investigated. After deletion of the M2‐receptor the maximal inhibition by carbachol was significantly reduced (by 41–72%), but not abolished, in all tissues. After deletion of the M4‐receptor a moderate and significant reduction of the maximal inhibition by carbachol (by 28%) was observed only in the vas deferens. Experiments with the muscarinic antagonists methoctramine and pirenzepine confirmed that the presynaptic muscarinic receptors were predominantly M2 in atria and bladder and probably a mixture of M2 and M4 in the vas deferens. Experiments in the urinary bladder with the cholinesterase inhibitor physostigmine and the muscarinic antagonist ipratropium demonstrated that endogenously released acetylcholine predominantly acted through M2‐receptors to inhibit noradrenaline release. However, the results do not exclude a minor contribution of M4‐receptors to this endogenous inhibition. In conclusion, our results clearly indicate that the release‐inhibiting muscarinic receptors on postganglionic sympathetic axons in mouse atria, bladder and vas deferens represent mixtures of M2‐ and non‐M2‐receptors. The non‐M2‐receptors remain unknown in atria and the bladder, and may represent primarily M4‐receptors in the vas deferens. These results reveal an unexpected heterogeneity among the muscarinic receptors mediating inhibition of noradrenaline release.

Crosstalk between presynaptic angiotensin receptors, bradykinin receptors and α2‐autoreceptors in sympathetic neurons: a study in α2‐adrenoceptor‐deficient mice

In mouse atria, angiotensin II and bradykinin lose much or all of their noradrenaline release‐enhancing effect when presynaptic α2‐autoinhibition does not operate either because of stimulation with very brief pulse trains or because of treatment with α2 antagonists. We now studied this operational condition in α2‐adrenoceptor‐deficient mice. Release of 3H‐noradrenaline was elicited by electrical stimulation. In tissues from wild‐type (WT) mice, angiotensin II and bradykinin increased the overflow of tritium evoked by 120 pulses at 3 Hz. This enhancement did not occur or was much reduced when tissues were stimulated by 120 pulses at 3 Hz in the presence of rauwolscine and phentolamine, or when they were stimulated by 20 pulses at 50 Hz. In tissues from mice lacking the α2A‐adrenoceptor (α2AKO) or the α2B‐adrenoceptor (α2BKO), the concentration–response curves of angiotensin II and bradykinin (120 pulses at 3 Hz) were unchanged. In tissues from mice lacking the α2C‐adrenoceptor (α2CKO) or both the α2A‐ and the α2C‐adrenoceptor (α2ACKO), the concentration–response curves were shifted to the same extent downwards. As in WT tissues, angiotensin II and bradykinin lost most or all of their effect in α2AKO and α2ACKO tissues when rauwolscine and phentolamine were present or trains consisted of 20 pulses at 50 Hz. Rauwolscine and phentolamine increased tritium overflow evoked by 120 pulses at 3 Hz up to seven‐fold in WT and α2BKO tissues, three‐fold in α2AKO and α2CKO tissues, and two‐fold in α2ACKO tissues. Results confirm that angiotensin II and bradykinin require ongoing α2‐autoinhibition for the full extent of their release‐enhancing effect. Specifically, they require ongoing α2C‐autoinhibition. The peptide effects that remain in α2C‐autoreceptor‐deficient mice seem to be because of α2B‐autoinhibition. The results hence also suggest that in addition to α2A‐ and α2C‐ mouse postganglionic sympathetic neurons possess α2B‐autoreceptors.