Experimental Physiology

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Alterations in Notch signalling in skeletal muscles from mdx and dko dystrophic mice and patients with Duchenne muscular dystrophy

01 April 2014
New Findings

  • What is the central question of this study?

    The Notch signalling pathway plays an important role in muscle regeneration, and activation of the pathway has been shown to enhance muscle regeneration in aged mice. It is unknown whether Notch activation will have a similarly beneficial effect on muscle regeneration in the context of Duchenne muscular dystrophy (DMD).

  • What is the main finding and its importance?

    Although expression of Notch signalling components is altered in both mouse models of DMD and in human DMD patients, activation of the Notch signalling pathway does not confer any functional benefit on muscles from dystrophic mice, suggesting that other signalling pathways may be more fruitful targets for manipulation in treating DMD.

  • Abstract

    In Duchenne muscular dystrophy (DMD), muscle damage and impaired regeneration lead to progressive muscle wasting, weakness and premature death. The Notch signalling pathway represents a central regulator of gene expression and is critical for cellular proliferation, differentiation and apoptotic signalling during all stages of embryonic muscle development. Notch activation improves muscle regeneration in aged mice, but its potential to restore regeneration and function in muscular dystrophy is unknown. We performed a comprehensive examination of several genes involved in Notch signalling in muscles from dystrophin-deficient mdx and dko (utrophin- and dystrophin-null) mice and DMD patients. A reduction of Notch1 and Hes1 mRNA in tibialis anterior muscles of dko mice and quadriceps muscles of DMD patients and a reduction of Hes1 mRNA in the diaphragm of the mdx mice were observed, with other targets being inconsistent across species. Activation and inhibition of Notch signalling, followed by measures of muscle regeneration and function, were performed in the mouse models of DMD. Notch activation had no effect on functional regeneration in C57BL/10, mdx or dko mice. Notch inhibition significantly depressed the frequency–force relationship in regenerating muscles of C57BL/10 and mdx mice after injury, indicating reduced force at each stimulation frequency, but enhanced the frequency–force relationship in muscles from dko mice. We conclude that while Notch inhibition produces slight functional defects in dystrophic muscle, Notch activation does not significantly improve muscle regeneration in murine models of muscular dystrophy. Furthermore, the inconsistent expression of Notch targets between murine models and DMD patients suggests caution when making interspecies comparisons.

    Reactive oxygen species mediated diaphragm fatigue in a rat model of chronic intermittent hypoxia

    01 April 2014

    Respiratory muscle dysfunction documented in sleep apnoea patients is perhaps due to oxidative stress secondary to chronic intermittent hypoxia (CIH). We sought to explore the effects of different CIH protocols on respiratory muscle form and function in a rodent model. Adult male Wistar rats were exposed to CIH (n = 32) consisting of 90 s normoxia–90 s hypoxia (either 10 or 5% oxygen at the nadir; arterial O2 saturation ~90 or 80%, respectively] for 8 h per day or to sham treatment (air–air, n = 32) for 1 or 2 weeks. Three additional groups of CIH-treated rats (5% O2 for 2 weeks) had free access to water containing N-acetyl cysteine (1% NAC, n = 8), tempol (1 mm, n = 8) or apocynin (2 mm, n = 8). Functional properties of the diaphragm muscle were examined ex vivo at 35°C. The myosin heavy chain and sarco(endo)plasmic reticulum Ca2+-ATPase isoform distribution, succinate dehydrogenase and glyercol phosphate dehydrogenase enzyme activities, Na+–K+-ATPase pump content, concentration of thiobarbituric acid reactive substances, DNA oxidation and antioxidant capacity were determined. Chronic intermittent hypoxia (5% oxygen at the nadir; 2 weeks) decreased diaphragm muscle force and endurance. All three drugs reversed the deleterious effects of CIH on diaphragm endurance, but only NAC prevented CIH-induced diaphragm weakness. Chronic intermittent hypoxia increased diaphragm muscle myosin heavy chain 2B areal density and oxidized glutathione/reduced glutathione (GSSG/GSH) ratio. We conclude that CIH-induced diaphragm dysfunction is reactive oxygen species dependent. N-Acetyl cysteine was most effective in reversing CIH-induced effects on diaphragm. Our results suggest that respiratory muscle dysfunction in sleep apnoea may be the result of oxidative stress and, as such, antioxidant treatment could prove a useful adjunctive therapy for the disorder.

    The possible mechanisms by which maternal hypothyroidism impairs insulin secretion in adult male offspring in rats

    01 April 2014

    Previous studies have recently shown that maternal hypothyroidism leads to impaired glucose metabolism and reduced insulin secretion in adult offspring in rats. The aim of this study was to locate the defect in the insulin secretion pathway induced by maternal hypothyroidism. Pregnant Wistar rats were divided into two groups; the control group consumed water, while the hypothyroid (FH) group received water containing 0.025% 6-propyl-2-thiouracil during gestation. An intravenous glucose tolerance test was carried out on 5-month-old male offspring. In in vitro studies, the effects of various secretagogues and inhibitors acting at different levels of the insulin secretion cascade were investigated, and insulin content, insulin secretion and glucokinase activity of the islets were compared. Although insulin content of the FH islets did not differ from that of control islets, insulin secretion from FH islets was reduced when it was challenged by glucose or arginine. Compared with control islets, activities of both hexokinase and glucokinase were also significantly decreased in the FH islets. Although, in both groups, increasing glibenclamide and nifedipine concentrations in the presence of 16.7 mmol l–1 glucose increased and decreased insulin secretion, respectively, the percentage of changes in secretion of FH islets was significantly lower compared with control islets. The response of FH islets to high extracellular potassium concentration and diazoxide was also significantly lower than that of the control islets. These findings demonstrate that impaired insulin secretion in the FH group is probably related to alterations in different steps of the insulin secretion pathway and not in the insulin pool of β-cells.

    The combined influence of fat consumption and repeated mental stress on brachial artery flow-mediated dilatation: a preliminary study

    01 April 2014

    Experienced separately, both acute mental stress and high-fat meal consumption can transiently impair endothelial function, and the purpose of the present study was to investigate their combined impact. On four separate days, 10 healthy men (23 years old) underwent brachial artery flow-mediated dilatation (FMD) tests, before and hourly for 4 h post-consumption of a high-fat (HFM; 54 g fat) or low-fat meal (LFM; 0 g fat; each meal ~1000 calories), with hourly mental stress (mental arithmetic, speech) or control (counting) tasks (conditions HFM+S, LFM+S, HFM and LFM). Data are presented as means ± SD. Plasma triglycerides increased and remained elevated after the high-fat but not the low-fat meal (P = 0.004) and were not affected by mental stress (P = 0.329). Indices of stress reactivity increased during mental stress tasks (mean arterial pressure, ~20 mmHg; heart rate, ~22 beats min–1; salivary cortisol, ~2.37 nmol l–1; and plasma noradrenaline, ~0.17 ng ml–1) and were not influenced by meal (P > 0.05). There was no effect of the type of meal on FMD (P = 0.562); however, FMD was 4.5 ± 0.5% in the control conditions and 5.8 ± 0.6% in the mental stress conditions (P = 0.087), and this difference was significant when normalized for the shear stress stimulus (FMD/area under the curve of shear stress, P = 0.045). Overall, these preliminary data suggest that postprandial FMD was augmented with mental stress irrespective of meal type. These results are contrary to previous reports of impaired endothelial function after mental stress or fat consumption independently and highlight the need to further investigate the mechanisms underlying the interactions between these factors.

    Effect of acute moderate exercise on induced inflammation and arterial function in older adults

    01 April 2014

    Acute inflammation reduces flow-mediated vasodilatation and increases arterial stiffness in young healthy individuals. However, this response has not been studied in older adults. The aim of this study, therefore, was to evaluate the effect of acute induced systemic inflammation on endothelial function and wave reflection in older adults. Furthermore, an acute bout of moderate-intensity aerobic exercise can be anti-inflammatory. Taken together, we tested the hypothesis that acute moderate-intensity endurance exercise, immediately preceding induced inflammation, would be protective against the negative effects of acute systemic inflammation on vascular function. Fifty-nine healthy volunteers between 55 and 75 years of age were randomized to an exercise or a control group. Both groups received a vaccine (induced inflammation) and sham (saline) injection in a counterbalanced crossover design. Inflammatory markers, endothelial function (flow-mediated vasodilatation) and measures of wave reflection and arterial stiffness were evaluated at baseline and at 24 and 48 h after injections. There were no significant differences in endothelial function and arterial stiffness between the exercise and control group after induced inflammation. The groups were then analysed together, and we found significant differences in the inflammatory markers 24 and 48 h after induction of acute inflammation compared with sham injection. However, flow-mediated vasodilatation, augmentation index normalized for heart rate (AIx75) and β-stiffness did not change significantly. Our results suggest that acute inflammation induced by influenza vaccination did not affect endothelial function in older adults.

    Corrigendum

    01 April 2014

    The rhythms of life: what your body clock means to you!

    01 April 2014

    Until we turned our nights into days and began to travel in aircraft across multiple time zones, we were largely unaware that we possess a ‘day within’ driven by an internal body clock. Yet the striking impairment of our abilities in the early hours of the morning soon reminds us that we are slaves to our biology. Our ability to perform mathematical calculations or other intellectual tasks between 04.00 and 06.00 h is worse than if we had consumed several shots of whisky and would be classified as legally drunk. Biological clocks drive or alter our sleep patterns, alertness, mood, physical strength, blood pressure and every other aspect of our physiology and behaviour. Our emerging understanding of how these 24 h rhythms are generated and regulated is not only one of the great success stories of modern biology, but is also informing many areas of human health. Sleep and circadian rhythm disruption (SCRD) is a feature shared by some of the most challenging diseases of our time, including neuropsychiatric illness and serious disorders of the eye. Sleep and circadian rhythm disruption is also commonly seen across many sectors of society, from teenagers to shift workers. We also now appreciate that SCRD is far more than feeling sleepy at an inappropriate time. It promotes multiple illnesses ranging across abnormal metabolism, heart disease, reduced immunity, increased stress and abnormal cognition and mood states. This short review considers how 24 h rhythms are generated and regulated, the consequences of working against our body clock and the emerging relationship between SCRD and mental illness.

    Pathophysiology of human heart failure: importance of skeletal muscle myopathy and reflexes

    01 April 2014

    In the last 20 years there has been mounting evidence that chronic heart failure (CHF) has a complex pathophysiology, which begins with an abnormality of the heart as a ‘primum movens’, but involves adaptive changes in many body parts, including the cardiovascular, musculoskeletal, renal, neuroendocrine, haemostatic, immune and inflammatory systems. Alterations in skeletal muscle are also of importance in limiting functional capacity in patients with CHF, because reduced physical activity plays some part in the muscle alterations in CHF. On the whole, these abnormalities resemble those induced by physical deconditioning. Moreover, the overactivation of signals originating from skeletal muscle receptors (mechano-metaboreceptors) is an intriguing hypothesis proposed to explain the origin of symptoms and the beneficial effect of exercise training in the CHF syndrome. These reflexes may contribute to sympathetic overactivation, to exercise intolerance and to the progression of CHF syndrome. The so-called metaboreflex has been reported to be hyperactive in CHF and to be responsible for a paradoxical increase in systemic vascular resistance and decrease in cardiac output whenever activated in these patients. This report is a brief summary of the latest news in this area of research.

    Editorial Board

    01 March 2014

    Memory consolidation in humans: new evidence and opportunities

    01 March 2014

    We are endlessly fascinated by memory; we desire to improve it and fear its loss. While it has long been recognized that brain regions such as the hippocampus are vital for supporting memories of our past experiences (autobiographical memories), we still lack fundamental knowledge about the mechanisms involved. This is because the study of specific neural signatures of autobiographical memories in vivo in humans presents a significant challenge. However, recent developments in high-resolution structural and functional magnetic resonance imaging coupled with advanced analytical methods now permit access to the neural substrates of memory representations that has hitherto been precluded in humans. Here, I describe how the application of ‘decoding’ techniques to brain-imaging data is beginning to disclose how individual autobiographical memory representations evolve over time, deepening our understanding of systems-level consolidation. In particular, this prompts new questions about the roles of the hippocampus and ventromedial prefrontal cortex and offers new opportunities to interrogate the elusive memory trace that has for so long confounded neuroscientists.

    The role of brain natriuretic peptide in maintaining myometrial quiescence during pregnancy

    01 March 2014

    Myometrial quiescence is a physiological stage of the myometrium during pregnancy. It is a period of active relaxation of the myometrial smooth muscle cells; myometrial quiescence is responsible for maintaining pregnancy. The precise mechanisms underlying myometrial quiescence have not been completely elucidated, although many mediators and cellular pathways have been described as playing a role. Fetal membranes (chorion and amnion) produce and release one or more substances that inhibit myometrial contractions, playing a central role in the maintenance of myometrial quiescence. Brain natriuretic peptide (BNP) is more potent than any other natriuretic peptide in inhibiting myometrial contractions in vitro. Brain natriuretic peptide is produced by the chorion and amnion, mainly during myometrial quiescence, and decreasing towards the end of pregnancy. Production of BNP is reduced in fetal membranes obtained from women in preterm labour. It is postulated that BNP, acting in a paracrine fashion, plays a key role in the maintaining myometrial quiescence and, therefore, controlling the duration of pregnancy. Furthermore, it is postulated that a premature decrease of BNP production by the fetal membranes may cause preterm labour and preterm birth.

    Myometrial physiology - time to translate?

    01 March 2014

    In this short review, we discuss how recent insights into myometrial physiology may be taken forward and translated into much-needed novel therapies for problems associated with labour. We consider excitation–contraction coupling in the myometrium and how this relates to our understanding of the changes that occur to produce myometrial contractions and successful labour. We then discuss how this information has already been used in the development of drugs to either stimulate or relax the myometrium, to address the needs of women with either slow (dystocic) labours or threatened preterm labours, respectively. We next present the data showing how basic physiological findings pertaining to hypoxia and lactate production have been taken and translated into a tool for predicting and thus better managing difficult labours. We then highlight examples of where physiological research has started to provide mechanistic insight into clinical problems associated with labour and parturition (obesity, diabetes, advanced maternal age, postdate and twin pregnancies) and suggest how these findings could be translated into new therapies for difficult labours.

    Kv7 and Kv11 channels in myometrial regulation

    01 March 2014

    Ion channels play a key role in defining myometrial contractility. Modulation of ion channel populations is proposed to underpin gestational changes in uterine contractility associated with the transition from uterine quiescence to active labour. Of the myriad ion channels present in the uterus, this article will focus upon potassium channels encoded by the KCNQ genes and ether-à-go-go-related (ERG) genes. Voltage-gated potassium channels encoded by KCNQ and ERG (termed Kv7 and Kv11, respectively) are accepted as major determinants of neuronal excitability and the duration of the cardiac action potential. However, there is now growing appreciation that these ion channels have a major functional impact in vascular and non-vascular smooth muscle. Moreover, Kv7 channels may be potential therapeutic targets for the treatment of preterm labour.

    Assessment of myometrial transcriptome changes associated with spontaneous human labour by high-throughput RNA-seq

    01 March 2014

    The transition of the human uterus from a quiescent to a contractile state takes place over a number of weeks. On such biological time scales, cellular phenotype is modified by changes in the transcriptome, which in turn is under the control of the underlying endocrine, paracrine, and biophysical processes resulting from the ongoing pregnancy. In this study, we characterize the transition of the human myometrial transcriptome at term from not in labour (NIL) to in labour (LAB) using high throughput RNA sequencing (RNA-seq). RNA was isolated from the myometrium of uterine biopsies from patients at term who were not in labour (n = 5) and at term in spontaneous labour (n = 5) without augmentation. A total of 143.6 million separate reads were sequenced, achieving, on average, ~13 times coverage of the expressed human transcriptome per sample. Principal component analysis indicated that the NIL and LAB transcriptomes could be distinguished as two distinct clusters. A comparison of the NIL and LAB groups, using three different statistical approaches (baySeq, edgeR, and DESeq), demonstrated an overlap of 764 differentially expressed genes. A comparison with currently available microarray data revealed only a partial overlap in differentially expressed genes. We conclude that the described RNA-seq data sets represent the first fully annotated catalogue of expressed mRNAs in human myometrium. When considered together, the full expression repertoire and the differentially expressed gene sets should provide an excellent resource for formulating new hypotheses of physiological function, as well as the discovery of novel therapeutic targets.

    The importance of the smooth muscle cytoskeleton to preterm labour

    01 March 2014

    Multiple mechanisms have been shown to regulate the onset of labour in a co-operative and complex manner. One factor, myometrial stretch and associated increases in wall tension, has been implicated clinically in the initiation of labour and especially the aetiology of preterm labour. Recent work on the mechanisms involved has led to the finding that the intracellular Ca2+ requirement for activation of the myometrial contractile filaments increases during gestation. The decreased Ca2+ sensitivity correlates with an increase in the expression of caldesmon, an actin-binding protein and inhibitor of myosin activation, during pregnancy. In late pregnancy, an increase in extracellular signal-regulated kinase-mediated caldesmon phosphorylation occurs, which appears to reverse the inhibitory action of caldesmon during labour. Force generated by the myometrial contractile filaments is communicated across the plasmalemma to the uterine wall through focal adhesions. Phospho-tyrosine screening and mass spectrometry of stretched myometrial samples identified several stretch-activated focal adhesion proteins. This Src-mediated focal adhesion signalling appears to provide a tunable, i.e. regulated, tension sensor and force transmitter in the myometrial cell. In other parallel studies, biophysical measurements of smooth muscle compliance at both the cellular and tissue levels suggest that decreases in cellular compliance due to changing interactions of the actin cytoskeleton with the focal adhesions may also promote increases in uterine wall tension. These results, taken together, suggest that focal adhesion proteins and their interaction with the cytoskeleton may present a new mode of regulation of uterine contractility.

    Finding new agents in medicinal plants to act on the myometrium

    01 March 2014

    This report summarizes work investigating the effects of some medicinal plants on uterine contraction. As there is a clinical need to find better drugs to help control uterine activity, and novel compounds are sought, the mechanisms whereby the medicinal plants exert their effects, as well as their major compounds, are discussed. By identifying the plants, major constituents and mechanisms, this review also illustrates the potential for development of new drugs, so that better ways to treat uterine disorders will be available to women worldwide.

    Elevated levels of branched-chain amino acids have little effect on pancreatic islet cells, but L-arginine impairs function through activation of the endoplasmic reticulum stress response

    01 March 2014

    Recent metabolic profiling studies have identified a correlation between branched-chain amino acid levels, insulin resistance associated with prediabetes and susceptibility to type 2 diabetes. Glucose and lipids in chronic excess have been reported to induce toxic effects in pancreatic β-cells, but the effect of elevated amino acid concentrations on primary islet cell function has not been investigated to date. The aim of this study was to investigate the effect of chronic exposure to various amino acids on islet cell function in vitro. Isolated rat islets were incubated over periods of 48 h with a range of concentrations of individual amino acids (0.1 μm to 10 mm). After 48 h, islets were assessed for glucose-dependent insulin secretion capacity, proliferation or islet cell apoptosis. We report that elevated levels of branched-chain amino acids have little effect on pancreatic islet cell function or viability; however, increased levels of the amino acid l-arginine were found to be β-cell toxic, causing a dose-dependent decrease in insulin secretion accompanied by a decrease in islet cell proliferation and an increase in islet cell apoptosis. These effects were not due to l-arginine-dependent increases in production of nitric oxide but arose through elicitation of the islet cell endoplasmic reticulum stress response. This novel finding indicates, for the first time, that the l-arginine concentration in vitro may impact negatively on islet cell function, thus indicating further complexity in relationship to in vivo susceptibility of β-cells to nutrient-induced dysfunction.