Feasibility, Efficacy, and Safety of Percutaneous Muscle Biopsies in Patients With Chronic Liver Disease.

INTRODUCTION: Sarcopenia is present in many chronic disease states including decompensated end stage liver disease (ESLD) and non-cirrhotic non-alcoholic fatty liver disease (NAFLD). Sarcopenia in ESLD can negatively impact quality of life and increase mortality. Despite this, very little is understood about the mechanisms of sarcopenia in these conditions. One key reason for this is the reluctance to undertake percutaneous muscle biopsies due to the perceived increased risks. ESLD can induce thrombocytopaenia and coagulopathy which significantly increases the risk of bleeding. In addition, patients with either NAFLD or ESLD often have co-morbidities that would require additional care and risk assessment. Thus, the aim of this study was to establish an effective and safe protocol for the implementation of percutaneous muscle biopsies in patients with NAFLD and ESLD. METHODS: A total of 47 patients with ESLD and 9 patients with non-cirrhotic NAFLD were recruited from the Liver Unit, Queen Elizabeth Hospital (Birmingham, United Kingdom). A total of 71 percutaneous vastus lateralis biopsies were attempted over two study visits. A vigorous safety screening occurred prior to and during each visit and a strict protocol was followed to mitigate against complications and risk. RESULTS: A total of 85% of patients consented to the muscle biopsy at either visit (48/56). A total of 9% of consented biopsies could not occur due to medical considerations, including high international normalised ratio (INR) (n = 3) and the use of aspirin (n = 4). Muscle tissue was obtained from 90% of attempts, with a mean average yield (wet weight tissue) of 98.1 ± 52.9 mg. CONCLUSION: Percutaneous muscle biopsies are both feasible and yield sufficient tissue in an ESLD population. The procedure is effective for obtaining muscle tissue whilst also safe, with only one adverse event. This study provides evidence for the successful use of muscle biopsies in this population, even in consideration of disease specific complications, medications, and comorbidities.

Protocol for understanding acute sarcopenia: a cohort study to characterise changes in muscle quantity and physical function in older adults following hospitalisation.

BACKGROUND: Older adults are vulnerable to the effects of acute sarcopenia (acute muscle insufficiency) following hospitalisation. However, this condition remains poorly characterised to date. It is hypothesised that acute sarcopenia arises due to a combination of bed rest and inflammatory surge. This study aims to characterise changes in muscle quantity and function, determining which factors (clinical and biological) are most predictive, and how these relate to change in physical function at 13 weeks. METHODS: This study will include three groups of patients aged 70 years and older; patients undergoing elective colorectal surgery, patients admitted for emergency abdominal surgery, and patients admitted under general medicine with acute bacterial infections. Changes in muscle quantity (Bilateral Anterior Thigh Thickness with ultrasound and bioelectrical impedance analysis) and muscle function (muscle strength, physical performance) within 1 week of hospitalisation or surgery will be characterised, with follow-up of patients at 13 weeks. Physical function will be measured using the Patient Reported Outcome Measures Information System, and the Short Physical Performance Battery (or gait speed alone within 1 week of surgery). DISCUSSION: This study will fully characterise changes in muscle quantity and function in hospitalised older adults and enable risk stratification towards targeted interventions in clinical practice. The results of this study will inform further research involving interventions to ameliorate changes. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03858192 ; Prospectively registered 28th February 2019.

Frailty Levels In Geriatric Hospital paTients (FLIGHT)-the prevalence of frailty among geriatric populations within hospital ward settings: a systematic review protocol.

INTRODUCTION: Frailty is a common and clinically significant condition in geriatric populations, associated with adverse health outcomes such as hospitalisation, disability and mortality. Although there are systematic reviews/meta-analyses assessing the prevalence of frailty in community-dwelling older adults, nursing home residents, and cancer and general surgery patients, there are none assessing the overall prevalence of frailty in geriatric hospital inpatients. METHODS AND ANALYSIS: This review will systematically search and analyse the prevalence of frailty within geriatric hospital inpatients within the literature. A search will be employed on the platforms of Ovid, Web of Science and databases of Cumulative Index to Nursing and Allied Health Literature (CINAHL) Plus, SCOPUS and the Cochrane Library. Any observational or experimental study design which utilises a validated operational definition of frailty, reports the prevalence of frailty, has a minimum age ≥65 years, attempts to assess the whole ward/clinical population and occurs in hospital inpatients, will be included. Title and abstract and full-text screenings will be conducted by three reviewers. Methodological quality of eligible studies will be assessed using the Joanna Briggs Institute critical appraisal tool. Data extraction will be performed by two reviewers. If sufficient data are available, a meta-analysis synthesising pooled estimates of the prevalence of frailty and pre-frailty, as well as the prevalence of frailty stratified by age, sex, operational frailty definition, prevalent morbidities, ward type and location, among older hospitalised inpatients will be conducted. Clinical heterogeneity will be assessed by two reviewers. Statistical heterogeneity will be assessed through a Cochran Q test, and an I 2 test performed to assess its magnitude. ETHICS AND DISSEMINATION: Ethical approval was not required as primary data will not be collected. Findings will be disseminated through publication in peer reviewed open access scientific journals, public engagement events, conference presentations and social media. PROSPERO REGISTRATION NUMBER: 79202.

A pilot observational study measuring acute sarcopenia in older colorectal surgery patients.

OBJECTIVE: To explore variability in acute changes in muscle mass and function in older patients undergoing elective colorectal surgery, as well as feasibility of measures, in order to refine study processes to inform the protocol for a larger study. RESULTS: Results are presented for seven participants recruited to this pilot study. It is possible to perform serial measurements of bilateral anterior thigh thickness (BATT) and handgrip strength prior to, within 24 h of surgery, and 1 week postoperatively. Gait speed can be reliably measured preoperatively and at 1 week postoperatively. In this pilot study, BATT and gait speed declined at 1 week postoperatively (median BATT 4.17 cm, 3.47 cm, p = 0.028; median gait speed 0.89 m/s, 0.83 m/s, p = 0.043). Baseline hsCRP correlated with change in BATT (τb = 0.73, p = 0.04) and baseline DHEA-S correlated with change in gait speed (τb = 0.87, p = 0.02). This pilot study has assisted to refine the protocol for our larger study, which will further characterise these changes.

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

BACKGROUND: 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. METHODS: Biomarkers of multiple molecular pathways associated with muscle regeneration were analysed using quantitative polymerase chain reaction in quadriceps muscle samples obtained from healthy elderly sarcopenic (HSE, n = 7) or non-sarcopenic (HENS, n = 21) and healthy middle-aged control (HMC, n = 22) groups. An in vitro system of myogenesis (using myoblasts from human donors aged 17-83 years) was used to mimic the environmental challenges of muscle regeneration over time. RESULTS: The muscle biopsies showed evidence of satellite cell activation in HENS (Pax3, P < 0.01, Pax7, P < 0.0001) compared with HMC. Early myogenesis markers Myogenic Differentiation 1 (MyoD1) and Myogenic factor 5 (Myf5) (P < 0.0001) and the late myogenesis marker myogenin (MyoG) (P < 0.01) were increased in HENS. In addition, there was a 30-fold upregulation of TNF-α in HENS compared with HMC (P < 0.0001). The in vitro system demonstrated age-related upregulation of pro-inflammatory cytokines (2-fold upregulation of interleukin (IL)-6, IL-8 mRNA, increased secretion of tumor necrosis factor-α (TNF-α) and IL-6, all P < 0.05) associated with impaired kinetics of myotube differentiation. The HSE biopsy samples showed satellite cell activation (Pax7, P < 0.05) compared with HMC. However, no significant upregulation of the early myogenesis (MyoD and Myf5) markers was evident; only the late myogenesis marker myogenin was upregulated (P < 0.05). Higher activation of the oxidative stress pathway was found in HENS compared with the HSE group. In contrast, there was 10-fold higher upregulation of HSPA1A a stress-induced chaperone acting upon misfolded proteins in HSE compared with the HENS group. CONCLUSIONS: Both pathological and adaptive processes are active in skeletal muscle during healthy ageing. Muscle regeneration pathways are activated during healthy ageing, but there is evidence of dysregulation in sarcopenia. In addition, increased cellular stress, with an impaired oxidative-stress and mis-folded protein response (HSPA1A), may be associated with the development of sarcopenia. The in vitro system of young and old myoblasts replicated some of the differences between young and old muscle.

Comprehensive proteome analysis of human skeletal muscle in cachexia and sarcopenia: a pilot study.

BACKGROUND: Cancer cachexia (cancer-induced muscle wasting) is found in a subgroup of cancer patients leaving the patients with a poor prognosis for survival due to a lower tolerance of the chemotherapeutic drug. The cause of the muscle wasting in these patients is not fully understood, and no predictive biomarker exists to identify these patients early on. Skeletal muscle loss is an inevitable consequence of advancing age. As cancer frequently occurs in old age, identifying and differentiating the molecular mechanisms mediating muscle wasting in cancer cachexia vs. age-related sarcopenia are a challenge. However, the ability to distinguish between them is critical for early intervention, and simple measures of body weight may not be sufficiently sensitive to detect cachexia early. METHODS: We used a range of omics approaches: (i) undepleted proteome was quantified using advanced high mass accuracy mass spectrometers in SWATH-MS acquisition mode; (ii) phospho epitopes were quantified using protein arrays; and (iii) morphology was assessed using fluorescent microscopy. RESULTS: We quantified the soluble proteome of muscle biopsies from cancer cachexia patients and compared them with cohorts of cancer patients and healthy individuals with and without age-related muscle loss (aka age-related sarcopenia). Comparing the proteomes of these cohorts, we quantified changes in muscle contractile myosins and energy metabolism allowing for a clear identification of cachexia patients. In an in vitro time lapse experiment, we mimicked cancer cachexia and identified signal transduction pathways governing cell fusion to play a pivotal role in preventing muscle regeneration. CONCLUSIONS: The work presented here lays the foundation for further understanding of muscle wasting diseases and holds the promise of overcoming ambiguous weight loss as a measure for defining cachexia to be replaced by a precise protein signature.

Sex differences in the effect of fish-oil supplementation on the adaptive response to resistance exercise training in older people: a randomized controlled trial.

BACKGROUND: Resistance exercise increases muscle mass and function in older adults, but responses are attenuated compared with younger people. Data suggest that long-chain n-3 polyunsaturated fatty acids (PUFAs) may enhance adaptations to resistance exercise in older women. To our knowledge, this possibility has not been investigated in men. OBJECTIVE: We sought to determine the effects of long-chain n-3 PUFA supplementation on resistance exercise training-induced increases in muscle mass and function and whether these effects differ between older men and women. DESIGN: Fifty men and women [men: n = 27, mean ± SD age: 70.6 ± 4.5 y, mean ± SD body mass index (BMI; in kg/m2): 25.6 ± 4.2; women: n = 23, mean ± SD age: 70.7 ± 3.3 y, mean ± SD BMI: 25.3 ± 4.7] were randomly assigned to either long-chain n-3 PUFA (n = 23; 3 g fish oil/d) or placebo (n = 27; 3 g safflower oil/d) and participated in lower-limb resistance exercise training twice weekly for 18 wk. Muscle size, strength, and quality (strength per unit muscle area), functional abilities, and circulating metabolic and inflammatory markers were measured before and after the intervention. RESULTS: Maximal isometric torque increased after exercise training to a greater (P < 0.05) extent in the long-chain n-3 PUFA group than in the placebo group in women, with no differences (P > 0.05) between groups in men. In both sexes, the effect of exercise training on maximal isokinetic torque at 30, 90, and 240° s-1, 4-m walk time, chair-rise time, muscle anatomic cross-sectional area, and muscle fat did not differ (P > 0.05) between groups. There was a greater (P < 0.05) increase in muscle quality in women after exercise training in the long-chain n-3 PUFA group than in the placebo group, with no such differences in men (P > 0.05). Long-chain n-3 PUFAs resulted in a greater decrease (P < 0.05) than the placebo in plasma triglyceride concentrations in both sexes, with no differences (P > 0.05) in glucose, insulin, or inflammatory markers. CONCLUSION: Long-chain n-3 PUFA supplementation augments increases in muscle function and quality in older women but not in older men after resistance exercise training. This trial was registered at clinicaltrials.gov as NCT02843009.

Loss of oxidative defense and potential blockade of satellite cell maturation in the skeletal muscle of patients with cancer but not in the healthy elderly.

Muscle wasting in old age or cancer may result from failed myofiber regeneration and/or accelerated atrophy. This study aimed to determine from transcriptomic analysis of human muscle the integrity of the cellular stress response system in relation to satellite cell differentiation or apoptosis in patients with cancer (weight-stable (CWS) or weight-losing (CWL)) or healthy elderly (HE) when compared with healthy middle-aged controls (HMA). 28 patients with cancer (CWS: 18 and CWL: 10), HE: 21 and HMA: 20 underwent biopsy of quadriceps muscle. The expression of transcription factors for muscle regeneration (Pax3, Pax7 and MyoD) was increased in CWS and HE compared with HMA (p≤0.001). In contrast, the expression of the late myogenic differentiation marker MyoG was reduced in CWS and CWL but increased in HE (p≤0.0001). Bax was significantly increased in CWS, CWL and HE (p≤0.0001). Expression of the oxidative defense genes SOD2, GCLM, and Nrf2 was decreased in CWS and CWL but increased in HE (p≤0.0001). There is evidence for blockade of satellite cell maturation, upregulation of apoptosis and reduced oxidative defense in the muscle of cancer patients. In the healthy elderly the potential for differentiation and oxidative defense is maintained.

Evaluating potential biomarkers of cachexia and survival in skeletal muscle of upper gastrointestinal cancer patients.

BACKGROUND: In order to grow the potential therapeutic armamentarium in the cachexia domain of supportive oncology, there is a pressing need to develop suitable biomarkers and potential drug targets. This pilot study evaluated several potential candidate biomarkers in skeletal muscle biopsies from a cohort of upper gastrointestinal cancer (UGIC) patients. METHODS: One hundred seven patients (15 weight-stable healthy controls (HC) and 92 UGIC patients) were recruited. Mean (standard deviation) weight-loss of UGIC patients was 8.1 (9.3%). Cachexia was defined as weight-loss ≥5%. Rectus abdominis muscle was obtained at surgery and was analysed by western blotting or quantitative real-time-polymerase chain reaction. Candidate markers were selected according to previous literature and included Akt and phosphorylated Akt (pAkt, n = 52), forkhead box O transcription factors (n = 59), ubiquitin E3 ligases (n = 59, control of muscle anabolism/catabolism), BNIP3 and GABARAPL1 (n = 59, as markers of autophagy), myosin heavy-chain (MyHC, n = 54), dystrophin (n = 39), ß-dystroglycan (n = 52), and ß-sarcoglycan (n = 52, as markers of structural alteration in a muscle). Patients were followed up for an average of 1255 days (range 581-1955 days) or until death. Patients were grouped accordingly and analysed by (i) all cancer patients vs. HC; (ii) cachectic vs. non-cachectic cancer patients; and (iii) cancer patients surviving ≤1 vs. >1 year post operatively. RESULTS: Cancer compared with HC patients had reduced mean (standard deviation) total Akt protein [0.49 (0.31) vs. 0.89 (0.17), P = 0.001], increased ratio of phosphorylated to total Akt [1.33 (1.04) vs. 0.32 (0.21), P = 0.002] and increased expression of GABARAPL1 [1.60 (0.76) vs. 1.10 (0.57), P = 0.024]. ß-Dystroglycan levels were higher in cachectic compared with non-cachectic cancer patients [1.01 (0.16) vs. 0.87 (0.20), P = 0.007]. Survival was shortened in patients with low compared with high MyHC levels (median 316 vs. 1326 days, P = 0.023) and dystrophin levels (median 341 vs. 660 days, P = 0.008). CONCLUSIONS: The present study has identified intramuscular protein level of ß-dystroglycan as a potential biomarker of cancer cachexia. Changes in the structural elements of muscle (MyHC or dystrophin) appear to be survival biomarkers.

Suppression of skeletal muscle turnover in cancer cachexia: evidence from the transcriptome in sequential human muscle biopsies.

PURPOSE: The mechanisms underlying muscle wasting in patients with cancer remain poorly understood, and consequently there remains an unmet clinical need for new biomarkers and treatment strategies. EXPERIMENTAL DESIGN: Microarrays were used to examine the transcriptome in single biopsies from healthy controls (n = 6) and in paired biopsies [pre-resection baseline (weight-loss 7%) and 8 month post-resection follow-up (disease-free/weight-stable for previous 2 months)] from quadriceps muscle of patients with upper gastrointestinal cancer (UGIC; n = 12). RESULTS: Before surgery, 1,868 genes were regulated compared with follow-up (false discovery rate, 6%). Ontology analysis showed that regulated genes belonged to both anabolic and catabolic biologic processes with overwhelming downregulation in baseline samples. No literature-derived genes from preclinical cancer cachexia models showed higher expression in baseline muscle. Comparison with healthy control muscle (n = 6) revealed that despite differences in the transcriptome at baseline (941 genes regulated), the muscle of patients at follow-up was similar to control muscle (2 genes regulated). Physical activity (step count per day) did not differ between the baseline and follow-up periods (P = 0.9), indicating that gene expression differences reflected the removal of the cancer rather than altered physical activity levels. Comparative gene expression analysis using exercise training signatures supported this interpretation. CONCLUSIONS: Metabolic and protein turnover-related pathways are suppressed in weight-losing patients with UGIC whereas removal of the cancer appears to facilitate a return to a healthy state, independent of changes in the level of physical activity.

Sexual dimorphism modulates the impact of cancer cachexia on lower limb muscle mass and function.

BACKGROUND & AIMS: There is a sparsity of data on the impact of cachexia on human muscle function. This study examined the relationship between cachexia, quality of life and the mass/function/mechanical quality of lower limb skeletal muscle in gastrointestinal cancer patients. METHODS: Quadriceps strength and lower limb power were measured in 54 patients with gastrointestinal cancer (n = 24 ≥ 10% weight-loss) and 18 healthy controls. Quadriceps cross-sectional area was measured in 33/54 patients and in all controls using MRI. Muscle mechanical quality was defined as quadriceps strength/unit quadriceps cross-sectional area. Quality of life was assessed using the EORTC QLQ-C30. Patients with weight-loss ≥ 10% were classified as cachectic. RESULTS: In male cachectic patients, quadriceps strength (p = 0.003), lower limb power (p = 0.026), quadriceps cross-sectional area (p = 0.019) and muscle quality (p = 0.008) were reduced compared with controls. In female cachectic patients, quadriceps strength (p = 0.001) and muscle quality (p = 0.001) were reduced compared with controls. Physical function (p = 0.013) and fatigue (p = 0.004) quality of life scores were reduced in male cachectic compared with non-cachectic patients, but not in females. CONCLUSIONS: Muscle quality is reduced in cancer patients. The degree of impairment of lower limb muscle mass, quality and function and the impact on quality of life varies with weight-loss and sex.

Intramyocellular lipid droplets increase with progression of cachexia in cancer patients.

BACKGROUND: Intramyocellular lipids are an important source of fuel for mitochondrial fat oxidation and play an important role in intramuscular lipid homeostasis. We hypothesised that due to the phenotype associated with cancer cachexia, there would exist an association between increasing weight loss and the number/size of intramyocellular lipid droplets. METHODS: Nineteen cancer patients and 6 controls undergoing surgery were recruited. A rectus abdominis biopsy was performed and processed for transmission electron microscopy (TEM). The number of intramyocellular lipid droplets and lipid droplet diameter were calculated from the TEM images. CT scans, performed as part of patients' routine care, were analysed to determine amount of adipose (intermuscular, visceral and subcutaneous) and muscle tissue. RESULTS: Compared with controls, cancer patients had increased numbers of lipid droplets (mean (SD) 1.8 (1.9) vs. 6.4 (9.1) per ×2,650 field, respectively, p = 0.036). Mean (SD) lipid droplet diameter was also higher in cancer patients compared with controls (0.42 (0.13) vs. 0.24 (0.21) µm, p = 0.015). Mean lipid droplet count correlated positively with the severity of weight loss (R = 0.51, p = 0.025) and negatively with CT-derived measures of intermuscular fat (R = -0.53, p = 0.022) and visceral fat (R = -0.51, p = 0.029). CONCLUSIONS: This study suggests that the number and size of intramyocellular lipid droplets is increased in the presence of cancer and increases further with weight loss/loss of adipose mass in other body compartments.

Magnetic resonance imaging with k-means clustering objectively measures whole muscle volume compartments in sarcopenia/cancer cachexia.

BACKGROUND & AIMS: Sarcopenia and cachexia are characterized by infiltration of non-contractile tissue within muscle which influences area and volume measurements. We applied a statistical clustering (k-means) technique to magnetic resonance (MR) images of the quadriceps of young and elderly healthy women and women with cancer to objectively separate the contractile and non-contractile tissue compartments. METHODS: MR scans of the thigh were obtained for 34 women (n = 16 young, (median) age 26 y; n = 9 older, age 80 y; n = 9 upper gastrointestinal cancer patients, age 65 y). Segmented regions of consecutive axial images were used to calculate cross-sectional area and (gross) volume. The k-means unsupervised algorithm was subsequently applied to the MR binary mask image array data with resultant volumes compared between groups. RESULTS: Older women and women with cancer had 37% and 48% less quadriceps muscle respectively than young women (p < 0.001). Application of k-means subtracted a significant 9%, 14% and 20% non-contractile tissue from the quadriceps of young, older and patient groups respectively (p < 0.001). There was a significant effect of group (i.e., cancer vs healthy) when controlling for age as a covariate (p = 0.003). CONCLUSIONS: K-means objectively separates contractile and non-contractile tissue components. Women with upper GI cancer have significant fatty infiltration throughout whole muscle groups which is maintained when controlling for age.

Skeletal muscle IGF-I isoform expression in healthy women after isometric exercise.

CONTEXT: Little is known about the expression of skeletal muscle-specific isoforms of the insulin-like growth factor-I (IGF-I) gene in response to exercise in humans. Data for women are sparse and the influences of age and physical training status are unknown. Here we aimed to describe the expression of mRNA for the IGF-I isoforms in the quadriceps muscles of women at rest and after isometric exercise. OBJECTIVE: To categorize mRNA expression of the IGF-I splice variants IGF-IEa and IGF-IEc (MGF) in healthy women, determine whether isometric exercise stimulates expression, and to determine the duration of the response. DESIGN: Observational study. SETTING: Clinical research facility. PARTICIPANTS: Six healthy women (median age 38 years, range 25-43 years). INTERVENTION: Single bout of maximal isometric knee extension undertaken as 20 sets of 6 contractions. Muscle samples from the lateral mass of the quadriceps were obtained by needle biopsy at baseline, 2.5h and 50h after exercise. MAIN OUTCOME MEASURES: Quantification of mRNAs for IGF-IEa and MGF. RESULTS: mRNA for the IGF-IEa transcript was significantly elevated by 50h post-exercise compared to baseline (p=0.005) and mRNA for MGF was significantly elevated by 2.5h (p=0.026). There were no statistically significant differences between measurements at 2.5h and 50h for either transcript. CONCLUSIONS: We have shown that the upregulation of the muscle specific IGF-IEa and MGF isoforms occurs within 2.5h of a single bout of isometric exercise in women. The upregulation persists for at least 2 days after exercise. We have also demonstrated a potentially safe and effective way of studying the responsiveness of these isoforms to resistance exercise in future studies of older and/or frail individuals.