Smoke-induced neuromuscular junction degeneration precedes the fibre type shift and atrophy in chronic obstructive pulmonary disease.

KEY POINTS: Chronic obstructive pulmonary disease (COPD) is largely caused by smoking, and patient limb muscle exhibits a fast fibre shift and atrophy. We show that this fast fibre shift is associated with type grouping, suggesting recurring cycles of denervation-reinnervation underlie the type shift. Compared to patients with normal fat-free mass index (FFMI), patients with low FFMI exhibited an exacerbated fibre type shift, marked accumulation of very small persistently denervated muscle fibres, and a blunted denervation-responsive transcript profile, suggesting failed denervation precipitates muscle atrophy in patients with low FFMI. Sixteen weeks of passive tobacco smoke exposure in mice caused neuromuscular junction degeneration, consistent with a key role for smoke exposure in initiating denervation in COPD. ABSTRACT: A neurological basis for the fast fibre shift and atrophy seen in limb muscle of patients with chronic obstructive pulmonary disease (COPD) has not been considered previously. The objective of our study was: (1) to determine if denervation contributes to fast fibre shift and muscle atrophy in COPD; and (2) to assess using a preclinical smoking mouse model whether chronic tobacco smoke (TS) exposure could initiate denervation by causing neuromuscular junction (NMJ) degeneration. Vastus lateralis muscle biopsies were obtained from severe COPD patients [n = 10 with low fat-free mass index (FFMI), 65 years; n = 15 normal FFMI, 65 years) and healthy age- and activity-matched non-smoker control subjects (CON; n = 11, 67 years), to evaluate morphological and transcriptional markers of denervation. To evaluate the potential for chronic TS exposure to initiate these changes, we examined NMJ morphology in male adult mice following 16 weeks of passive TS exposure. We observed a high proportion of grouped fast fibres and a denervation transcript profile in COPD patients, suggesting that motor unit remodelling drives the fast fibre type shift in COPD patient limb muscle. A further exacerbation of fast fibre grouping in patients with low FFMI, coupled with blunted reinnervation signals, accumulation of very small non-specific esterase hyperactive fibres and neural cell adhesion molecule-positive type I and type II fibres, suggests denervation-induced exhaustion of reinnervation contributes to muscle atrophy in COPD. Evidence from a smoking mouse model showed significant NMJ degeneration, suggesting that recurring denervation in COPD is probably caused by decades of chronic TS exposure.

Autophagy in locomotor muscles of patients with chronic obstructive pulmonary disease.

RATIONALE: Locomotor muscle atrophy develops in patients with chronic obstructive pulmonary disease (COPD) partly because of increased protein degradation by the ubiquitin-proteasome system. It is not known if autophagy also contributes to protein degradation. OBJECTIVES: To investigate whether autophagy is enhanced in locomotor muscles of stable patients with COPD, to quantify autophagy-related gene expression in these muscles, and to identify mechanisms of autophagy induction. METHODS: Muscle biopsies were obtained from two cohorts of control subjects and patients with COPD and the numbers of autophagosomes in the vastus lateralis and tibialis anterior muscles, the levels of LC3B protein lipidation, and the expression of autophagy-related genes were measured in the vastus lateralis muscle. To investigate potential pathways that might induce the activation of autophagy, measures were taken of protein kinase B (AKT), mTORC1, and AMPK pathway activation, transcription factor regulation, proteasome activation, and oxidative stress. MEASUREMENTS AND MAIN RESULTS: Autophagy is enhanced in the locomotor muscles of patients with COPD as shown by significantly higher numbers of autophagosomes in affected muscles as compared with control subjects. Autophagosome number inversely correlates with FEV1. In the vastus lateralis, LC3B protein lipidation is increased by COPD and the expression of autophagy-related gene expressions is up-regulated. LC3B lipidation inversely correlates with thigh cross-sectional area, FEV1, and FEV1/FVC ratio. Enhanced autophagy is associated with activation of the AMPK pathway and FOXO transcription factors, inhibition of the mTORC1 and AKT pathways, and the development of oxidative stress. CONCLUSIONS: Autophagy is significantly enhanced in locomotor muscles of stable patients with COPD. The degree of autophagy correlates with severity of muscle atrophy and lung function impairment.

The feasibility and acceptability of neuromuscular electrical stimulation to improve exercise performance in patients with advanced cancer: a pilot study.

BACKGROUND: To determine the feasibility and acceptability of lower limb neuromuscular electrical stimulation (NMES) as a home-based exercise therapy in patients with cancer who could not attend hospital-based exercise training. METHODS: A single-arm prospective pilot study of NMES, applied daily to both quadriceps muscles for six weeks. Participants were recruited from patients referred to a hospital-based multi-disciplinary supportive care team specializing in treatment of patients with nutritional depletion and functional decline. RESULTS: Of the 15 participants who underwent baseline testing, 10 (67%) completed the study and only one (7%) withdrew because of discomfort due to NMES treatment. 7/10 (70%) of participants used NMES at least three times a week for the duration of the study. Use of NMES did not lead to significant improvements in physical performance tests. CONCLUSIONS: NMES is a feasible and acceptable intervention for home use in patients with cancer, poor performance status and metastatic disease. However, whether NMES is an effective strategy to stabilize or improve physical performance in such patients is not proven.

Measurement of CYP1A2 and CYP3A4 activity by a simplified Geneva cocktail approach in a cohort of free-living individuals: a pilot study.

Introduction: The cytochrome P450 enzyme subfamilies, including CYP3A4 and CYP1A2, have a major role in metabolism of a range of drugs including several anti-cancer treatments. Many factors including environmental exposures, diet, diseaserelated systemic inflammation and certain genetic polymorphisms can impact the activity level of these enzymes. As a result, the net activity of each enzyme subfamily can vary widely between individuals and in the same individual over time. This variability has potential major implications for treatment efficacy and risk of drug toxicity, but currently no assays are available for routine use to guide clinical decision-making. Methods: To address this, a mass spectrometry-based method to measure activities of CYP3A4, CYP1A2 was adapted and tested in free-living participants. The assay results were compared with the predicted activity of each enzyme, based on a self-report tool capturing diet, medication, chronic disease state, and tobacco usage. In addition, a feasibility test was performed using a low-volume dried blood spots (DBS) on two different filter-paper supports, to determine if the same assay could be deployed without the need for repeated standard blood tests. Results: The results confirmed the methodology is safe and feasible to perform in free-living participants using midazolam and caffeine as test substrates for CYP3A4 and CYP1A2 respectively. Furthermore, though similar methods were previously shown to be compatible with the DBS format, the assay can also be performed successfully while incorporating glucuronidase treatment into the DBS approach. The measured CYP3A4 activity score varied 2.6-fold across participants and correlated with predicted activity score obtained with the self-report tool. The measured CYP1A2 activity varied 3.5-fold between participants but no correlation with predicted activity from the self-report tool was found. Discussion: The results confirm the wide variation in CYP activity between individuals and the important role of diet and other exposures in determining CYP3A4 activity. This methodology shows great potential and future cross-sectional and longitudinal studies using DBS are warranted to determine how best to use the assay results to guide drug treatments.

Weight changes correlate with alterations in subjective physical function in advanced cancer patients referred to a specialized nutrition and rehabilitation team.

PURPOSE: The aim of this study is to assess whether short-term weight gain correlates with improvements in subjective markers of quality of life and physical function in patients referred to a clinic for management of cancer cachexia. METHODS: A retrospective review of the records of 306 patients referred to a specialized multi-disciplinary supportive care team with particular interest in treating cancer cachexia. Weight changes between each of the first three clinic visits, were correlated with the corresponding changes in patient-rated performance status, perceived strength and quality of life. In a second cohort of 56 patients, the correlation between perceived strength and quality of life was re-tested using a more detailed quality of life tool. RESULTS: Even over short time intervals positive correlations were observed for weight change vs. change in patient-rated performance status (Rs > 0.15, P < 0.05), and for changes in perceived strength vs. quality of life (Rs > 0.33, P < 0.001). The correlation between changes in patient-rated strength and quality of life was consistent across all subgroups studied and was reproducible when using a different, validated, quality of life tool (FAACT) in a second independent patient cohort. CONCLUSIONS: Weight gains are associated with subjective improvements in physical functioning, and changes in perceived physical strength are consistently correlated with quality of life.

Chronic aryl hydrocarbon receptor activity phenocopies smoking-induced skeletal muscle impairment.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) patients exhibit skeletal muscle atrophy, denervation, and reduced mitochondrial oxidative capacity. Whilst chronic tobacco smoke exposure is implicated in COPD muscle impairment, the mechanisms involved are ambiguous. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that activates detoxifying pathways with numerous exogenous ligands, including tobacco smoke. Whereas transient AHR activation is adaptive, chronic activation can be toxic. On this basis, we tested the hypothesis that chronic smoke-induced AHR activation causes adverse muscle impact. METHODS: We used clinical patient muscle samples, and in vitro (C2C12 myotubes) and in vivo models (mouse), to perform gene expression, mitochondrial function, muscle and neuromuscular junction morphology, and genetic manipulations (adeno-associated virus-mediated gene transfer). RESULTS: Sixteen weeks of tobacco smoke exposure in mice caused muscle atrophy, neuromuscular junction degeneration, and reduced oxidative capacity. Similarly, smoke exposure reprogrammed the muscle transcriptome, with down-regulation of mitochondrial and neuromuscular junction genes. In mouse and human patient specimens, smoke exposure increased muscle AHR signalling. Mechanistically, experiments in cultured myotubes demonstrated that smoke condensate activated the AHR, caused mitochondrial impairments, and induced an AHR-dependent myotube atrophy. Finally, to isolate the role of AHR activity, expression of a constitutively active AHR mutant without smoke exposure caused atrophy and mitochondrial impairments in cultured myotubes, and muscle atrophy and neuromuscular junction degeneration in mice. CONCLUSIONS: These results establish that chronic AHR activity, as occurs in smokers, phenocopies the atrophy, mitochondrial impairment, and neuromuscular junction degeneration caused by chronic tobacco smoke exposure.

Proteolysis in illness-associated skeletal muscle atrophy: from pathways to networks.

Improvements in health in the past decades have resulted in increased numbers of the elderly in both developed and developing regions of the world. Advances in therapy have also increased the prevalence of patients with chronic and degenerative diseases. Muscle wasting, a feature of most chronic diseases, is prominent in the elderly and contributes to both morbidity and mortality. A major research goal has been to identify the proteolytic system(s) that is responsible for the degradation of proteins that occurs in muscle atrophy. Findings over the past 20 years have clearly confirmed an important role of the ubiquitin proteasome system in mediating muscle proteolysis, particularly that of myofibrillar proteins. However, recent observations have provided evidence that autophagy, calpains and caspases also contribute to the turnover of muscle proteins in catabolic states, and furthermore, that these diverse proteolytic systems interact with each other at various levels. Importantly, a number of intracellular signaling pathways such as the IGF1/AKT, myostatin/Smad, PGC1, cytokine/NFκB, and AMPK pathways are now known to interact and can regulate some of these proteolytic systems in a coordinated manner. A number of loss of function studies have identified promising therapeutic approaches to the prevention and treatment of wasting. However, additional biomarkers and other approaches to improve early identification of patients who would benefit from such treatment need to be developed. The current data suggests a network of interacting proteolytic and signaling pathways in muscle. Future studies are needed to improve understanding of the nature and control of these interactions and how they work to preserve muscle function under various states of growth and atrophy.

Diagnostic criteria for cancer cachexia: reduced food intake and inflammation predict weight loss and survival in an international, multi-cohort analysis.

BACKGROUND: Cancer-associated weight loss (WL) associates with increased mortality. International consensus suggests that WL is driven by a variable combination of reduced food intake and/or altered metabolism, the latter often represented by the inflammatory biomarker C-reactive protein (CRP). We aggregated data from Canadian and European research studies to evaluate the associations of reduced food intake and CRP with cancer-associated WL (primary endpoint) and overall survival (OS, secondary endpoint). METHODS: The data set included a total of 12,253 patients at risk for cancer-associated WL. Patient-reported WL history (% in 6 months) and food intake (normal, moderately, or severely reduced) were measured in all patients; CRP (mg/L) and OS were measured in N = 4960 and N = 9952 patients, respectively. All measures were from a baseline assessment. Clinical variables potentially associated with WL and overall survival (OS) including age, sex, cancer diagnosis, disease stage, and performance status were evaluated using multinomial logistic regression MLR and Cox proportional hazards models, respectively. RESULTS: Patients had a mean weight change of -7.3% (±7.1), which was categorized as: ±2.4% (stable weight; 30.4%), 2.5-5.9% (19.7%), 6.0-10.0% (23.2%), 11.0-14.9% (12.0%), ≥15.0% (14.6%). Normal food intake, moderately, and severely reduced food intake occurred in 37.9%, 42.8%, and 19.4%, respectively. In MLR, severe WL (≥15%) (vs. stable weight) was more likely (P < 0.0001) if food intake was moderately [OR 6.28, 95% confidence interval (CI 5.28-7.47)] or severely reduced [OR 18.98 (95% CI 15.30-23.56)]. In subset analysis, adjusted for food intake, CRP was independently associated (P < 0.0001) with ≥15% WL [CRP 10-100 mg/L: OR 2.00, (95% CI 1.58-2.53)] and [CRP > 100 mg/L: OR 2.30 (95% CI 1.62-3.26)]. Diagnosis, stage, and performance status, but not age or sex, were significantly associated with WL. Median OS was 9.9 months (95% CI 9.5-10.3), with median follow-up of 39.7 months (95% CI 38.8-40.6). Moderately and severely reduced food intake and CRP independently predicted OS (P < 0.0001). CONCLUSIONS: Modelling WL as the dependent variable is an approach that can help to identify clinical features and biomarkers associated with WL. Here, we identify criterion values for food intake impairment and CRP that may improve the diagnosis and classification of cancer-associated cachexia.

Defining barriers to implementation of nutritional advice in patients with cachexia.

BACKGROUND: Cancer cachexia is a multidimensional wasting syndrome and a reduced dietary intake is both common and strongly correlated with degree of weight loss. Many patients with cachexia do not achieve recommended dietary intake even after nutritional counselling. Prior reports suggest this is likely due to barrier symptoms, but other potential contributory factors have not been studied in detail. METHODS: Dietitian-assigned barriers to successful nutritional intervention were recorded at each visit in all patients attending a multidisciplinary clinic for management of cancer cachexia. The barriers were grouped into 15 categories and classified as either symptom-related or not symptom-related. In addition, symptom scores, dietary intake, and weight change were recorded. RESULTS: Data on 94 new patients showed that 89% of patients had at least one major barrier. Four of the five most common barriers and 65% of all barriers identified were not symptom-related. Over sequential visits the specific barrier(s) in any one patient changed approximately 50% of the time. However, the presence of barriers did not render patients refractory to nutritional intervention and with intervention from the CNR-JGH team, mean dietary intake increased significantly. CONCLUSIONS: In advanced cancer patients with cachexia, non-symptom-related barriers to nutritional intervention are more common than symptom-related. Barriers are dynamic, and repeated careful evaluation over time is required to achieve optimal impact with nutritional intervention in cancer cachexia. Members of the multidisciplinary team need appropriate expertise to address the barriers identified and achieve optimal results with nutritional intervention.

Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic diseases.

We previously identified a common set of genes, termed atrogenes, whose expression is coordinately induced or suppressed in muscle during systemic wasting states (fasting, cancer cachexia, renal failure, diabetes). To determine whether this transcriptional program also functions during atrophy resulting from loss of contractile activity and whether atrogene expression correlates with the rate of muscle weight loss, we used cDNA microarrays and RT-polymerase chain reaction to analyze changes in mRNA from rat gastrocnemius during disuse atrophy induced by denervation or spinal cord isolation. Three days after Den or SI, the rate of muscle weight loss was greatest, and 78% of the atrogenes identified during systemic catabolic states were induced or repressed. Of particular interest were the large inductions of key ubiquitin ligases, atrogin-1 (35- to 44-fold) and MuRF1 (12- to 22-fold), and the suppression of PGC-1alpha and PGC-1beta coactivators (15-fold). When atrophy slowed (day 14), the expression of 92% of these atrogenes returned toward basal levels. At 28 days, the atrophy-inducing transcription factor, FoxO1, was still induced and may be important in maintaining the "atrophied" state. Thus, 1) the atrophy associated with systemic catabolic states and following disuse involves similar transcriptional adaptations; and 2) disuse atrophy proceeds through multiple phases corresponding to rapidly atrophying and atrophied muscles that involve distinct transcriptional patterns.

Physiological culture conditions alter myotube morphology and responses to atrophy treatments: implications for in vitro research on muscle wasting.

Standard in vitro myotube culture conditions are nonphysiological and there is increasing evidence that this may distort adaptations to both catabolic and anabolic stimuli and hamper preclinical research into mechanisms and treatments for muscle atrophy in cancer and other chronic diseases. We tested a new model of myotube culture which mimics more accurately the basal conditions for muscle tissue in patients with chronic disease, such as cancer. Myotubes derived from C2C12 myoblasts, cultured under the modified conditions were thinner, more numerous, with more uniform morphology and an increased proportion of mature myotubes. Furthermore, modified conditions led to increased expression of mir-210-3p, genes related to slow-twitch, oxidative phenotype and resistance to commonly used experimental atrophy-inducing treatments. However, treatment with a combination of drugs used in anti-cancer treatment (doxorubicin and dexamethasone) under the modified culture conditions did lead to myotube atrophy which was only partially prevented by co-administration of curcumin. The results underline the importance and potential advantages of using physiological conditions for in vivo experiments investigating mechanisms of muscle atrophy and especially for preclinical screening of therapies for cancer-related muscle wasting.

A multidisciplinary rehabilitation programme for cancer cachexia improves quality of life.

OBJECTIVES: Patients with cancer cachexia have severely impaired quality of life (QoL). Multidisciplinary, multimodal treatment approaches have potential for stabilising weight and correcting other features of this syndrome, but the impact on QoL is unknown. METHODS: A retrospective analysis of QoL in patients with advanced cancer, referred for the management of cachexia by a specialised multidisciplinary clinic (The McGill Cancer Nutrition Rehabilitation Program clinic at the Jewish General Hospital (CNR-JGH)). QoL was assessed at visits 1-3 using a dedicated QoL tool for cachexia, and the change in QoL was calculated for each patient. The correlation between clinical features and QoL at baseline and subsequent change in QoL was analysed, to determine what factors predict improvements in QoL during the CNR-JGH intervention. RESULTS: 374 patients assessed at visit 1 with mean weight loss of 10.2% over the preceding 6 months. Baseline QoL scores were severely impaired but clinically important improvements were observed over visits 1-3 to the CNR-JGH clinic. Improvements in QoL were not determined by baseline characteristics and were similar in all patient subgroups. However, those patients who gained weight and increased their 6 min walk test (6MWT) had the greatest improvements in QoL. CONCLUSIONS: Improving management of all facets of the cancer cachexia syndrome, including poor QoL, remains a priority. The multimodal approach to management of cancer cachexia offered by the CNR-JGH results in clinically important improvements in QoL. All patients who are able to receive this type of intervention have similar potential to improve their QoL, but the greatest benefits are seen in those who gain weight and improve their 6MWT.

Eccentric Ergometer Training Promotes Locomotor Muscle Strength but Not Mitochondrial Adaptation in Patients with Severe Chronic Obstructive Pulmonary Disease.

Eccentric ergometer training (EET) is increasingly being proposed as a therapeutic strategy to improve skeletal muscle strength in various cardiorespiratory diseases, due to the principle that lengthening muscle actions lead to high force-generating capacity at low cardiopulmonary load. One clinical population that may particularly benefit from this strategy is chronic obstructive pulmonary disease (COPD), as ventilatory constraints and locomotor muscle dysfunction often limit efficacy of conventional exercise rehabilitation in patients with severe disease. While the feasibility of EET for COPD has been established, the nature and extent of adaptation within COPD muscle is unknown. The aim of this study was therefore to characterize the locomotor muscle adaptations to EET in patients with severe COPD, and compare them with adaptations gained through conventional concentric ergometer training (CET). Male patients were randomized to either EET (n = 8) or CET (n = 7) for 10 weeks and matched for heart rate intensity. EET patients trained on average at a workload that was three times that of CET, at a lower perception of leg fatigue and dyspnea. EET led to increases in isometric peak strength and relative thigh mass (p < 0.01) whereas CET had no such effect. However, EET did not result in fiber hypertrophy, as morphometric analysis of muscle biopsies showed no increase in mean fiber cross-sectional area (p = 0.82), with variability in the direction and magnitude of fiber-type responses (20% increase in Type 1, p = 0.18; 4% decrease in Type 2a, p = 0.37) compared to CET (26% increase in Type 1, p = 0.04; 15% increase in Type 2a, p = 0.09). EET had no impact on mitochondrial adaptation, as revealed by lack of change in markers of mitochondrial biogenesis, content and respiration, which contrasted to improvements (p < 0.05) within CET muscle. While future study is needed to more definitively determine the effects of EET on fiber hypertrophy and associated underlying molecular signaling pathways in COPD locomotor muscle, our findings promote the implementation of this strategy to improve muscle strength. Furthermore, contrasting mitochondrial adaptations suggest evaluation of a sequential paradigm of eccentric followed by concentric cycling as a means of augmenting the training response and attenuating skeletal muscle dysfunction in patients with advanced COPD.

Failed upregulation of TFAM protein and mitochondrial DNA in oxidatively deficient fibers of chronic obstructive pulmonary disease locomotor muscle.

BACKGROUND: Low mitochondrial content and oxidative capacity are well-established features of locomotor muscle dysfunction, a prevalent and debilitating systemic occurrence in patients with chronic obstructive pulmonary disease (COPD). Although the exact cause is not firmly established, physical inactivity and oxidative stress are among the proposed underlying mechanisms. Here, we assess the impact of COPD pathophysiology on mitochondrial DNA (mtDNA) integrity, biogenesis, and cellular oxidative capacity in locomotor muscle of COPD patients and healthy controls. We hypothesized that the high oxidative stress environment of COPD muscle would yield a higher presence of deletion-containing mtDNA and oxidative-deficient fibers and impaired capacity for mitochondrial biogenesis. METHODS: Vastus lateralis biopsies were analyzed from 29 COPD patients and 19 healthy age-matched controls for the presence of mtDNA deletions, levels of oxidatively damaged DNA, mtDNA copy number, and regulators of mitochondrial biogenesis as well the proportion of oxidative-deficient fibers (detected histologically as cytochrome c oxidase-deficient, succinate dehydrogenase positive (COX(-)/SDH(+) )). Additionally, mtDNA copy number and mitochondrial transcription factor A (TFAM) content were measured in laser captured COX(-)SDH(+) and normal single fibers of both COPD and controls. RESULTS: Compared to controls, COPD muscle exhibited significantly higher levels of oxidatively damaged DNA (8-hydroxy-2-deoxyguanosine (8-OHdG) levels = 387 ± 41 vs. 258 ± 21 pg/mL) and higher prevalence of mtDNA deletions (74 vs. 15 % of subjects in each group), which was accompanied by a higher abundance of oxidative-deficient fibers (8.0 ± 2.1 vs. 1.5 ± 0.4 %). Interestingly, COPD patients with mtDNA deletions had higher levels of 8-OHdG (457 ± 46 pg/mL) and longer smoking history (66.3 ± 7.5 years) than patients without deletions (197 ± 29 pg/mL; 38.0 ± 7.3 years). Transcript levels of regulators of mitochondrial biogenesis and oxidative metabolism were upregulated in COPD compared to controls. However, single fiber analyses of COX(-)/SDH(+) and normal fibers exposed an impairment in mitochondrial biogenesis in COPD; in healthy controls, we detected a marked upregulation of mtDNA copy number and TFAM protein in COX(-)/SDH(+) compared to normal fibers, reflecting the expected compensatory attempt by the oxidative-deficient cells to increase energy levels; in contrast, they were similar between COX(-)/SDH(+) and normal fibers in COPD patients. Taken together, these findings suggest that although the signaling factors regulating mitochondrial biogenesis are increased in COPD muscle, impairment in the translation of these signals prevents the restoration of normal oxidative capacity. CONCLUSIONS: Single fiber analyses provide the first substantive evidence that low muscle oxidative capacity in COPD cannot be explained by physical inactivity alone and is likely driven by the disease pathophysiology.

Patterns of gene expression in atrophying skeletal muscles: response to food deprivation.

During fasting and many systemic diseases, muscle undergoes rapid loss of protein and functional capacity. To define the transcriptional changes triggering muscle atrophy and energy conservation in fasting, we used cDNA microarrays to compare mRNAs from muscles of control and food-deprived mice. Expression of >94% of genes did not change, but interesting patterns emerged among genes that were differentially expressed: 1) mRNAs encoding polyubiquitin, ubiquitin extension proteins, and many (but not all) proteasome subunits increased, which presumably contributes to accelerated protein breakdown; 2) a dramatic increase in mRNA for the ubiquitin ligase, atrogin-1, but not most E3s; 3) a significant suppression of mRNA for myosin binding protein H (but not other myofibrillar proteins) and IGF binding protein 5, which may favor cell protein loss; 4) decreases in mRNAs for several glycolytic enzymes and phosphorylase kinase subunits, and dramatic increases in mRNAs for pyruvate dehydrogenase kinase 4 and glutamine synthase, which should promote glucose sparing and gluconeogenesis. During fasting, metallothionein mRNA increased dramatically, mRNAs for extracellular matrix components fell, and mRNAs that may favor cap-independent mRNA translation rose. Significant changes occurred in mRNAs for many growth-related proteins and transcriptional regulators. These transcriptional changes indicate a complex adaptive program that should favor protein degradation and suppress glucose oxidation in muscle. Similar analysis of muscles atrophying for other causes is allowing us to identify a set of atrophy-specific changes in gene expression.

Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression.

Skeletal muscle atrophy is a debilitating response to starvation and many systemic diseases including diabetes, cancer, and renal failure. We had proposed that a common set of transcriptional adaptations underlie the loss of muscle mass in these different states. To test this hypothesis, we used cDNA microarrays to compare the changes in content of specific mRNAs in muscles atrophying from different causes. We compared muscles from fasted mice, from rats with cancer cachexia, streptozotocin-induced diabetes mellitus, uremia induced by subtotal nephrectomy, and from pair-fed control rats. Although the content of >90% of mRNAs did not change, including those for the myofibrillar apparatus, we found a common set of genes (termed atrogins) that were induced or suppressed in muscles in these four catabolic states. Among the strongly induced genes were many involved in protein degradation, including polyubiquitins, Ub fusion proteins, the Ub ligases atrogin-1/MAFbx and MuRF-1, multiple but not all subunits of the 20S proteasome and its 19S regulator, and cathepsin L. Many genes required for ATP production and late steps in glycolysis were down-regulated, as were many transcripts for extracellular matrix proteins. Some genes not previously implicated in muscle atrophy were dramatically up-regulated (lipin, metallothionein, AMP deaminase, RNA helicase-related protein, TG interacting factor) and several growth-related mRNAs were down-regulated (P311, JUN, IGF-1-BP5). Thus, different types of muscle atrophy share a common transcriptional program that is activated in many systemic diseases.

Diagnostic criteria for the classification of cancer-associated weight loss.

PURPOSE: Existing definitions of clinically important weight loss (WL) in patients with cancer are unclear and heterogeneous and do not consider current trends toward obesity. METHODS: Canadian and European patients with cancer (n = 8,160) formed a population-based data set. Body mass index (BMI) and percent WL (%WL) were recorded, and patients were observed prospectively until death. Data were entered into a multivariable analysis controlling for age, sex, cancer site, stage, and performance status. Relationships for BMI and %WL to overall survival were examined to develop a grading system. RESULTS: Mean overall %WL was -9.7% ± 8.4% and BMI was 24.4 ± 5.1 kg/m(2), and both %WL and BMI independently predicted survival (P < .01). Differences in survival were observed across five categories of BMI (< 20.0, 20.0 to 21.9, 22.0 to 24.9, 25.0 to 27.9, and ≥ 28.0 kg/m(2); P < .001) and five categories of %WL (-2.5% to -5.9%, -6.0% to -10.9%, -11.0% to -14.9%, ≥ -15.0%, and weight stable (± 2.4%); P < .001). A 5 × 5 matrix representing the five %WL categories within each of the five BMI categories was graded based on median survival and prognostic significance. Weight-stable patients with BMI ≥ 25.0 kg/m(2) (grade 0) had the longest survival (20.9 months; 95% CI, 17.9 to 23.9 months), and %WL values associated with lowered categories of BMI were related to shorter survival (P < .001), as follows: grade 1, 14.6 months (95% CI, 12.9 to 16.2 months); grade 2, 10.8 months (95% CI, 9.7 to 11.9 months); grade 3, 7.6 months (95% CI, 7.0 to 8.2 months); and grade 4, 4.3 months (95% CI, 4.1 to 4.6 months). Survival discrimination by grade was observed within specific cancers, stages, ages, and performance status and in an independent validation sample (n = 2,963). CONCLUSION: A robust grading system incorporating the independent prognostic significance of both BMI and %WL was developed.

Anthracycline-containing chemotherapy causes long-term impairment of mitochondrial respiration and increased reactive oxygen species release in skeletal muscle.

Anticancer treatments for childhood acute lymphoblastic leukaemia (ALL) are highly effective but are now implicated in causing impaired muscle function in long-term survivors. However, no comprehensive assessment of skeletal muscle mitochondrial functions in long-term survivors has been performed and the presence of persistent chemotherapy-induced skeletal muscle mitochondrial dysfunction remains a strong possibility. Non-tumour-bearing mice were treated with two drugs that have been used frequently in ALL treatment (doxorubicin and dexamethasone) for up to 4 cycles at 3-week intervals and euthanized 3 months after the 4th cycle. Treated animals had impaired growth and lower muscle mass as well as reduced mitochondrial respiration and increased reactive oxygen species production per unit oxygen consumption. Mitochondrial DNA content and protein levels of key mitochondrial membrane proteins and markers of mitochondrial biogenesis were unchanged, but protein levels of Parkin were reduced. This suggests a novel pattern of chemotherapy-induced mitochondrial dysfunction in skeletal muscle that persists because of an acquired defect in mitophagy signaling. The results could explain the observed functional impairments in adult survivors of childhood ALL and may also be relevant to long-term survivors of other cancers treated with similar regimes.

After the chemotherapy: potential mechanisms for chemotherapy-induced delayed skeletal muscle dysfunction in survivors of acute lymphoblastic leukaemia in childhood.

There is evidence that survivors of childhood cancers, such as acute lymphoblastic leukemia (ALL), have increased rates of long-term skeletal muscle dysfunction. This places them at higher risk of physical restriction and functional impairment as well as potentially contributing to observed increases in cardiovascular disease and insulin resistance in later life. The mechanisms underlying these changes in skeletal muscle are unknown but chemotherapy drugs used in treatment for ALL are strongly implicated. Normal skeletal muscle growth, development, and function are dependent on correctly functioning muscle satellite cells, muscle motor neurons, and muscle mitochondria. Each of these key components is potentially susceptible to damage by chemotherapy in childhood, particularly prolonged courses including repeated administration of combination chemotherapy. If this chemotherapy-induced damage is not fully reversible, impairment of satellite cells, muscle motor innervation, and mitochondria could, either singly or together, lead to the emergence of delayed or persistent skeletal muscle dysfunction many years later. The known effects of individual drugs used in the treatment of ALL are outlined and the need for specific targeted studies to investigate the mechanisms underlying persistent muscle dysfunction in survivors of ALL and other childhood cancers is highlighted.