Angelika Meyer

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.

TAK-1/p38/nNFκB signaling inhibits myoblast differentiation by increasing levels of Activin A

BackgroundSkeletal-muscle differentiation is required for the regeneration of myofibers after injury. The differentiation capacity of satellite cells is impaired in settings of old age, which is at least one factor in the onset of sarcopenia, the age-related loss of skeletal-muscle mass and major cause of frailty. One important cause of impaired regeneration is increased levels of transforming growth factor (TGF)-β accompanied by reduced Notch signaling. Pro-inflammatory cytokines are also upregulated in aging, which led us hypothesize that they might potentially contribute to impaired regeneration in sarcopenia. Thus, in this study, we further analyzed the muscle differentiation-inhibition pathway mediated by pro-inflammatory cytokines in human skeletal muscle cells (HuSKMCs).MethodsWe studied the modulation of HuSKMC differentiation by the pro-inflammatory cytokines interleukin (IL)-1α and tumor necrosis factor (TNF)-α The grade of differentiation was determined by either imaging (fusion index) or creatine kinase (CK) activity, a marker of muscle differentiation. Secretion of TGF-β proteins during differentiation was assessed by using a TGF-β-responsive reporter-gene assay and further identified by means of pharmacological and genetic inhibitors. In addition, signaling events were monitored by western blotting and reverse transcription PCR, both in HuSKMC cultures and in samples from a rat sarcopenia study.ResultsThe pro-inflammatory cytokines IL-1α and TNF-α block differentiation of human myoblasts into myotubes. This anti-differentiation effect requires activation of TGF-β-activated kinase (TAK)-1. Using pharmacological and genetic inhibitors, the TAK-1 pathway could be traced to p38 and NFκB. Surprisingly, the anti-differentiation effect of the cytokines required the transcriptional upregulation of Activin A, which in turn acted through its established signaling pathway: ActRII/ALK/SMAD. Inhibition of Activin A signaling was able to rescue human myoblasts treated with IL-1β or TNF-α, resulting in normal differentiation into myotubes. Studies in aged rats as a model of sarcopenia confirmed that this pro-inflammatory cytokine pathway identified is activated during aging.ConclusionsIn this study, we found an unexpected connection between cytokine and Activin signaling, revealing a new mechanism by which cytokines affect skeletal muscle, and establishing the physiologic relevance of this pathway in the impaired regeneration seen in sarcopenia.

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.

Electrically evoked release of [3H]noradrenaline from mouse Cultured sympathetic neurons : Release-modulating heteroreceptors

Abstract: Cultured neurons from the thoracolumbar sympathetic chain of newborn mice are known to possess release‐inhibiting α2‐autoreceptors. The present study was carried out in a search for release‐modulating heteroreceptors on these neurons. Primary cultures were preincubated with [3H]noradrenaline and then superfused and stimulated by single pulses, trains of 8 pulses at 100 Hz, or trains of 36 pulses at 3 Hz. The cholinergic agonist carbachol reduced the evoked overflow of tritium. Experiments with antagonists indicated that the inhibition was mediated by M2 muscarinic receptors. The cannabinoid agonist WIN 55,212‐2 reduced the evoked overflow of tritium through CB1 receptors. Prostaglandin E2, sulprostone, and somatostatin also caused presynaptic inhibition. The inhibitory effects of carbachol, WIN 55,212‐2, prostaglandin E2, and somatostatin were abolished (at the highest concentration of WIN 55,212‐2 almost abolished) by pretreatment of the cultures with pertussis toxin (250 ng/ml). Several drugs, including the β2‐adrenoceptor agonist salbutamol, opioid receptor agonists, neuropeptide Y, angiotensin II, and bradykinin, failed to change the evoked overflow of tritium. These results demonstrate a distinct pattern of presynaptic inhibitory heteroreceptors, all coupled to pertussis toxin‐sensitive G proteins. The lack of operation of several presynaptic receptors known to exist in adult mice in situ may be due to the age of the (newborn) donor animals or to the culture conditions.

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.

Distinct mixtures of muscarinic receptor subtypes mediate inhibition of noradrenaline release in different mouse peripheral tissues, as studied with receptor knockout mice.

1 The muscarinic heteroreceptors modulating noradrenaline release in atria, urinary bladder and vas deferens were previously studied in mice in which the M2 or the M4 muscarinic receptor genes had been disrupted. These experiments showed that these tissues possessed both M2 and non‐M2 heteroreceptors. The analysis was now extended to mice in which either the M3, both the M2 and the M3, or both the M2 and the M4 genes had been disrupted (M3‐knockout, M2/3‐knockout and M2/4‐knockout). Tissues were preincubated with 3H‐noradrenaline and then stimulated electrically (20 pulses per 50 Hz). 2 In wild‐type atria, carbachol (0.01–100 μM) decreased the electrically evoked tritium overflow by maximally 60–78%. The maximum inhibition of carbachol was reduced to 57% in M3‐knockout and to 23% in M2/4‐knockout atria. Strikingly, the effect of carbachol was abolished in M2/3‐knockout atria. 3 In wild‐type bladder, carbachol (0.01–100 μM) reduced the evoked tritium overflow by maximally 57–71%. This effect remained unchanged in the M3‐knockout, but was abolished in the M2/4‐knockout bladder. 4 In wild‐type vas deferens, carbachol (0.01–100 μM) reduced the evoked tritium overflow by maximally 34–48%. The maximum inhibition of carbachol was reduced to 40% in the M3‐knockout and to 18% in the M2/4‐knockout vas deferens. 5 We conclude that the postganglionic sympathetic axons of mouse atria possess M2 and M3, those of the urinary bladder M2 and M4, and those of the vas deferens M2, M3 and M4 release‐inhibiting muscarinic receptors.

Short-term Low-Dose mTORC1 Inhibition in Aged Rats Counter-Regulates Age-Related Gene Changes and Blocks Age-Related Kidney Pathology

Abstract Rapalogs, inhibitors of mTORC1 (mammalian target of rapamycin complex 1), increase life span and delay age-related phenotypes in many species. However, the molecular mechanisms have not been fully elucidated. We determined gene expression changes comparing 6- and 24-month-old rats in the kidney, liver, and skeletal muscle, and asked which of these changes were counter-regulated by a clinically-translatable (short-term and low-concentration) treatment, with a rapalog (RAD001). Surprisingly, RAD001 had a more pronounced effect on the kidney under this regimen in comparison to the liver or skeletal muscle. Histologic evaluation of kidneys revealed that the severity of chronic progressive nephropathy lesions was lower in kidneys from 24-month-old rats treated with RAD001 compared with vehicle. In addition to other gene expression changes, c-Myc, which has been shown to regulate aging, was induced by aging in the kidney and counter-regulated by RAD001. RAD001 caused a decrease in c-Myc protein, which could be rescued by a proteasome inhibitor. These findings point to settings for use of mTORC1 inhibitors to treat age-related disorders, and highlight c-Myc regulation as one of the potential mechanisms by which mTORC1 inhibition is perturbing age-related phenotypes.

Alterations in the in vitro and in vivo regulation of muscle regeneration in healthy ageing and the influence of sarcopenia

Sarcopenia is defined as the age‐related loss of skeletal muscle mass and function. While all humans lose muscle with age, 2–5% of elderly adults develop functional consequences (disabilities). The aim of this study was to investigate muscle myogenesis in healthy elderly adults, with or without sarcopenia, compared with middle‐aged controls using both in vivo and in vitro approaches to explore potential biomarker or causative molecular pathways associated with sarcopenic versus non‐sarcopenic skeletal muscle phenotypes during ageing.