Kate A. Carey

NDRG2, a novel regulator of myoblast proliferation, is regulated by anabolic and catabolic factors

Skeletal muscle tissue undergoes adaptive changes in response to stress and the genes that control these processes are incompletely characterised. NDRG2 (N‐myc downstream‐regulated gene 2), a stress‐ and growth‐related gene, was investigated in skeletal muscle growth and adaption. While NDRG2 expression levels were found to be up‐regulated in both differentiated human and mouse myotubes compared with undifferentiated myoblasts, the suppression of NDRG2 in C2C12 myoblasts resulted in slowed myoblast proliferation. The increased expression levels of the cell cycle inhibitors, p21 Waf1/Cip1 and p27 Kip1, and of various muscle differentiation markers in NDRG2‐deficient myoblasts indicate that a lack of NDRG2 promoted cell cycle exiting and the onset of myogenesis. Furthermore, the analysis of NDRG2 regulation in C2C12 myotubes treated with catabolic and anabolic agents and in skeletal muscle from human subjects following resistance exercise training revealed NDRG2 gene expression to be down‐regulated during hypertrophic conditions, and conversely, up‐regulated during muscle atrophy. Together, these data demonstrate that NDRG2 expression is highly responsive to different stress conditions in skeletal muscle and suggest that the level of NDRG2 expression may be critical to myoblast growth and differentiation.

Activation of mTOR signalling in young and old human skeletal muscle in response to combined resistance exercise and whey protein ingestion

PURPOSE To investigate the impact of whey protein ingestion and resistance exercise training on the phosphorylation of mRNA translational signalling proteins in the skeletal muscle of young and old men. METHODS Sixteen healthy young (aged 18-25 years) and 15 healthy older men (aged 60-75 years) completed 12 weeks of resistance exercise and were randomly assigned to consume a whey protein (WPI) or placebo drink after each session. Muscle biopsies were collected before and 2 h after an acute exercise bout at the beginning and the end of training. RESULTS All subjects significantly increased strength after following strength training. Phosphorylation of mTOR was significantly greater in the WPI groups compared with placebo for both younger and older subjects. Phosphorylation of p70(S6K), eIF4G, and 4EBP1 was greater for older subjects consuming WPI. Phosphorylation of rpS6, eIF4G, and 4EBP1 tended to increase in the younger subjects that had consumed WPI. Post-training, younger subjects demonstrated a similar pattern of mTOR phosphorylation as seen pre-training. In contrast, the initial heightened phosphorylation of mTOR, p70(S6K), rpS6, and eIF4G in older muscle to combined resistance exercise and WPI ingestion became less pronounced after repeated training sessions. CONCLUSIONS In the untrained state, resistance exercise coupled with WPI increases the phosphorylation of proteins involved in mRNA translation compared with exercise alone. Post-training, WPI- and exercise-induced protein phosphorylation was reduced in older men, but not in younger men. Thus, strategies to induce hypertrophy should utilize protein and resistance training concurrently. Further investigations should delineate interventions that will maintain sensitivity to anabolic stimuli in older populations.

Exercise-induced activation of STAT3 signaling is increased with age.

Activation of the transcription factor signal transducers and activators of transcription (STAT) 3 is common to many inflammatory cytokines and growth factors, with recent evidence of involvement in skeletal muscle regeneration. The purpose of this study was to determine whether STAT3 signaling activation is regulated differentially, at rest and following intense resistance exercise, in aged human skeletal muscle. Skeletal muscle biopsies were harvested from healthy younger (n = 11, 20.4 +/- 0.8 years) and older men (n = 10, 67.4 +/- 1.3 years) under resting conditions and 2 h after the completion of resistance exercise. No differences were evident at rest, whereas the phosphorylation of STAT3 was significantly increased in old (23-fold) compared to young (5-fold) subjects after exercise. This correlated with significantly higher induction of the STAT3 target genes including; interleukin-6 (IL-6), JUNB, c-MYC, and suppressor of cytokine signaling (SOCS) 3 mRNA in older subjects following exercise. Despite increased SOCS3 mRNA, cellular protein abundance was suppressed. SOCS3 protein is an important negative regulator of STAT3 activation and cytokine signaling. Thus, in aged human muscle, elevated responsiveness of the STAT3 signaling pathway and suppressed SOCS3 protein are evident following resistance exercise. These data suggest that enhanced STAT3 signaling responsiveness to proinflammatory factors may impact on mechanisms of muscle repair and regeneration.

Plasma amino acid response after ingestion of different whey protein fractions

Background and objectives The digestion rate of proteins and subsequent absorption of amino acids can independently modulate protein metabolism. The objective of the present study was to examine the blood amino acid response to whey protein isolate (WPI), β-lactoglobulin-enriched WPI, hydrolysed WPI and a flavour-identical control. Methods Eight healthy adults (four female, four male) were recruited (mean±standard error of the mean: age, 27.0±0.76 years; body mass index, 23.2±0.8 kg/cm2) and after an overnight fast consumed 500 ml of each drink, each containing 25g protein, in a cross-over design. Blood was taken at rest and then every 15 min for 2 h post ingestion. Results Ingesting the β-lactoglobulin-enriched WPI drink resulted in significantly greater plasma leucine concentrations at 45–120 min and significantly greater branched-chain amino acid concentrations at 60–105 min post ingestion compared with hydrolysed WPI. No differences were observed between WPI and β-lactoglobulin-enriched WPI, and all protein drinks resulted in elevated blood amino acids compared with flavour-identical control. Conclusions In conclusion, whole proteins resulted in a more rapid absorption of leucine and branched-chain amino acid into the blood compared with the hydrolysed molecular form of whey protein.

Emerging therapies and challenges in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a hereditary neurodegenerative disease with severity ranging from progressive infantile paralysis and premature death (type I) to limited motor neuron loss and normal life expectancy (type IV). Without disease‐modifying therapies, the impact is profound for patients and their families. Improved understanding of the molecular basis of SMA, disease pathogenesis, natural history, and recognition of the impact of standardized care on outcomes has yielded progress toward the development of novel therapeutic strategies and are summarized. Therapeutic strategies in the pipeline are appraised, ranging from SMN1 gene replacement to modulation of SMN2 encoded transcripts, to neuroprotection, to an expanding repertoire of peripheral targets, including muscle. With the advent of preliminary trial data, it can be reasonably anticipated that the SMA treatment landscape will transform significantly. Advancement in presymptomatic diagnosis and screening programs will be critical, with pilot newborn screening studies underway to facilitate preclinical diagnosis. The development of disease‐modifying therapies will necessitate monitoring programs to determine the long‐term impact, careful evaluation of combined treatments, and further acceleration of improvements in supportive care. In advance of upcoming clinical trial results, we consider the challenges and controversies related to the implementation of novel therapies for all patients and set the scene as the field prepares to enter an era of novel therapies. Ann Neurol 2017;81:355–368

Actions of short-term fasting on human skeletal muscle myogenic and atrogenic gene expression

Background: Skeletal muscle mass is governed by multiple IGF-1-sensitive positive regulators of muscle-specific protein synthesis (myogenic regulatory factors which includes myoD, myogenin and Myf5) and negative regulators, including the atrogenic proteins myostatin, atrogin-1 and muscle ring finger 1 (MuRF-1). The coordinated control of these myogenic and atrogenic factors in human skeletal muscle following short-term fasting is currently unknown. Method: Healthy adults (n = 6, age 27.6 years) undertook a 40-hour fast. Skeletal muscle biopsy (vastus lateralis) and venous blood samples were taken 3, 15 and 40 h into the fast after an initial standard high-carbohydrate meal. Gene expression of the myogenic regulator factors (myoD, myogenin and Myf5) and the atrogenic factors (myostatin, atrogin-1 and MuRF-1) were determined by real-time PCR analysis. Plasma myostatin and IGF-1 were determined by ELISA. Results: There were no significant alterations in either the positive or negative regulators of muscle mass at either 15 or 40 h, when compared to gene expression measured 3 h after a meal. Similarly, plasma myostatin and IGF-1 were also unaltered at these times. Conclusions: Unlike previous observations in catabolic and cachexic diseased states, short-term fasting (40 h) fails to elicit marked alteration of the genes regulating both muscle-specific protein synthesis or atrophy. Greater periods of fasting may be required to initiate coordinated inhibition of myogenic and atrogenic gene expression.

Whey protein ingestion activates mTOR-dependent signalling after resistance exercise in young men: a double-blinded randomized controlled trial.

The effect of resistance exercise with the ingestion of supplementary protein on the activation of the mTOR cascade, in human skeletal muscle has not been fully elucidated. In this study, the impact of a single bout of resistance exercise, immediately followed by a single dose of whey protein isolate (WPI) or placebo supplement, on the activation of mTOR signalling was analyzed. Young untrained men completed a maximal single-legged knee extension exercise bout and were randomized to ingest either WPI supplement (n = 7) or the placebo (n = 7). Muscle biopsies were taken from the vastus lateralis before, and 2, 4 and 24 h post-exercise. WPI or placebo ingestion consumed immediately post-exercise had no impact on the phosphorylation of Akt (Ser473). However, WPI significantly enhanced phosphorylation of mTOR (Ser2448), 4E-BP1 (Thr37/46) and p70S6K (Thr389) at 2 h post-exercise. This study demonstrates that a single dose of WPI, when consumed in modest quantities, taken immediately after resistance exercise elicits an acute and transient activation of translation initiation within the exercised skeletal muscle.

Identification of novel genes expressed during rhabdomyosarcoma differentiation using cDNA microarrays.

Rhabdomyosarcomas (RMS) are highly aggressive tumors that are thought to arise as a consequence of the regulatory disruption of the growth and differentiation of skeletal muscle progenitor cells. Normal myogenesis is characterized by the expression of the myogenic regulatory factor gene family but, despite their expression in RMS, these tumor cells fail to complete the latter stages of myogenesis. The RMS cell line RD‐A was treated with 12‐O‐tetradecanoylphorbol‐13‐acetate to induce differentiation and cultured for 10 days. RNA was extracted on days 1, 3, 6, 8 and 10. A human skeletal muscle cDNA microarray was developed and used to analyze the global gene expression of RMS tumors over the time‐course of differentiation. As a comparison, the genes identified were subsequently examined during the differentiated primary human skeletal muscle cultures. Prothymosin alpha (PTMA), and translocase of inner mitochondrial membrane 10 (Tim10), two genes not previously implicated in RMS, showed reduced expression during differentiation. Marked differences in the expression of PTMA and Tim10 were observed during the differentiation of human primary skeletal muscle cells. These results identify several new genes with potential roles in the myogenic arrest present in rhabdomyosarcoma. PTMA expression in RMS biopsy samples might prove to be an effective diagnostic marker for this disease.

Nusinersen for SMA: expanded access programme

Background Spinal muscular atrophy (SMA) is a devastating motor neuron disorder causing progressive muscle weakness and respiratory insufficiency. We present the initial Australian experiences implementing the expanded access programme (EAP) to enable preapproval access to nusinersen, the first disease-modifying therapy, for SMA type 1. Methods An Australian multicentre, open-label EAP for nusinersen enrolled patients with infantile-onset SMA type 1 from November 2016 to September 2017. Standard-of-care medical therapy and treatment with intrathecal nusinersen were provided to all patients. Clinical and diagnostic characteristics, molecular genetics, treatment administered, and functional motor outcomes were assessed. Results A total of 20 patients with SMA type 1 met the inclusion criteria, of whom 16 consented and received nusinersen treatment. Median time to diagnosis from symptom onset was 5.0 months and was correlated with age of onset (r=0.54, P<0.05). Management shifts included proactive nutritional and pulmonary support in all newly diagnosed patients with increased complexity of decision making. Supplemental nutrition with or without nocturnal non-invasive ventilation was implemented during follow-up in new diagnoses with age of onset <3 months and 2 SMN2 copies. Conclusions The nusinersen EAP highlights difficulties in achieving early diagnosis and/or prevention, the evolution of optimal clinical care in a time of uncertain prognostication, resource implications and ethical issues in clinical practice for SMA type 1. These challenges are broadly relevant to the realisation of all novel therapeutics in neurological disorders.

Chemotherapy-Induced Peripheral Neuropathy in Long-term Survivors of Childhood Cancer: Clinical, Neurophysiological, Functional, and Patient-Reported Outcomes

Importance In light of the excellent long-term survival of childhood cancer patients, it is imperative to screen for factors affecting health, function, and quality of life in long-term survivors. Objective To comprehensively assess chemotherapy-induced peripheral neuropathy in childhood cancer survivors to define disease burden and functional effect and to inform screening recommendations. Design, Setting, and Participants In this cross-sectional observational study, cancer survivors who were treated with chemotherapy for extracranial malignancy before age 17 years were recruited consecutively between April 2015 and December 2016 from a single tertiary hospital-based comprehensive cancer survivorship clinic and compared with healthy age-matched controls. Investigators were blinded to the type of chemotherapy. A total of 169 patients met inclusion criteria, of whom 48 (28.4%) were unable to be contacted or declined participation. Exposures Chemotherapy agents known to be toxic to peripheral nerves. Main Outcomes and Measures The clinical peripheral neurological assessment using the Total Neuropathy Score was compared between recipients of different neurotoxic chemotherapy agents and control participants and was correlated with neurophysiological, functional, and patient-reported outcome measures. Results Of the 121 childhood cancer survivors included in this study, 65 (53.7%) were male, and the cohort underwent neurotoxicity assessments at a median (range) age of 16 (7-47) years, a median (range) 8.5 (1.5-29) years after treatment completion. Vinca alkaloids and platinum compounds were the main neurotoxic agents. Clinical abnormalities consistent with peripheral neuropathy were common, seen in 53 of 100 participants (53.0%) treated with neurotoxic chemotherapy (mean Total Neuropathy Score increase, 2.1; 95% CI, 1.4-2.9; P < .001), and were associated with lower limb predominant sensory axonal neuropathy (mean amplitude reduction, 5.8 &mgr;V; 95% CI, 2.8-8.8; P < .001). Functional deficits were seen in manual dexterity, distal sensation, and balance. Patient-reported outcomes demonstrating reduction in global quality of life and physical functioning were associated with the Total Neuropathy Score. Cisplatin produced long-term neurotoxicity more frequently than vinca alkaloids. Conclusions and Relevance Clinical abnormalities attributable to peripheral neuropathy were common in childhood cancer survivors and persisted long term, with concurrent deficits in patient-reported outcomes. Both the type of neurotoxic agent and a targeted clinical neurological assessment are important considerations when screening survivors for long-term neuropathy. Further development of peripheral neuropathy–specific pediatric assessment tools will aid research into neuroprotective and rehabilitative strategies.