AbstractBackgroundChronic kidney disease patients particularly with renal anaemia hyporesponsive to erythropoiesis‐stimulating agents (ESAs) are at a greater risk of having skeletal muscle mass (SMM) loss. Hypoxia‐inducible factor prolyl hydroxylase domain inhibitor (HIF‐PHI), a novel therapeutic agent for renal anaemia, potentially promotes angiogenesis, muscle repair, and homeostasis. However, effects of HIF‐PHIs on SMM remain unknown.MethodsThis retrospective observational cohort study enrolled 292 Japanese adults receiving maintenance haemodialysis at our dialysis centre. The dataset included 11 patients who received daprodustat for 6 months or longer during 1 December 2020 through 30 June 2022. From the previously published pooled cohort, we enrolled 281 participants from 1 August 2018 to 31 July 2019 prior to the approval of HIF‐PHIs for renal anaemia. SMM was assessed using modified creatinine index (mg/kg/day) calculated by age, sex, serum creatinine, and single‐pool Kt/V. Annual changes of SMM [ΔSMM (%)] were analysed with the least squares regression model and mixed‐effects model during 6‐ to 12‐month follow‐up period.ResultsThe median age of the participants was 63 years [interquartile range (IQR), 54–71 years], and 33% were female. The median ΔSMM levels (IQR) in the least squares regression model were 4.0% (−1.7% to 9.3%) in the HIF‐PHI group, 0.20% (−2.1% to 2.1%) in the no ESA group, and −0.94% (−3.0% to 1.3%) in the high ESA group using darbepoetin equivalent to 20 μg or more per week. Those in the mixed‐effects model were −1.7% (−1.2% to 3.8%), 0.09% (−1.4% to 1.3%), and −0.74% (−2.0% to 0.8%), respectively. The multivariable linear regression models revealed that HIF‐PHI use was associated with greater ΔSMM compared with the high ESA group [coefficient, 3.737; 95% confidence interval (CI), 1.216–6.258 in the least squares regression model or coefficient, 1.635; 95% CI, 0.068–3.201 in the mixed‐effects model, respectively].ConclusionsHIF‐PHI use led to greater ΔSMM in maintenance haemodialysis patients. HIF‐PHIs may minimize loss of SMM in patients with end‐stage kidney disease and renal anaemia.
RS reports grants from Japan Heart Foundation/Bayer Yakuhin Research Grant Abroad, during the conduct of the study. SvH has been a paid consultant for and/or received honoraria payments from AstraZeneca, Bayer, Boehringer Ingelheim, BRAHMS, Chugai, Grünenthal, Helsinn, Hexal, Novartis, Novo Nordisk, Pfizer, Pharmacosmos, Respicardia, Roche, Servier, Sorin, and Vifor and reports research support from Amgen, Boehringer Ingelheim, Pharmacosmos, IMI, and the German Centre for Cardiovascular Research (DZHK).
An emerging hypothesis is that the activation of innate immunity in the muscle of patients with chronic kidney disease (CKD) is implicated in the development and progression of wasting and cachexia. We previously observed that Toll-like receptor-4 (TLR4) and its downward NF-κB-dependent pro-inflammatory pathways are activated in CKD muscle. It is however unknown if TLR4 can activate the TLR4/NOD, LRR, and pyrin domain-containing protein 3 (NLRP3) inflammasome pathway, which is implicated in cardiovascular disease and frailty, clinical settings that are commonly observed in CKD patients. In a case–control cohort study, we hypothesized that a TLR4/NLRP3 inflammasome pathway is activated in skeletal muscle in uraemia. First, we studied the regulation TLR4/NLRP3/caspase-1 in skeletal muscle biopsies (20M/11F) obtained from 31 non-diabetic CKD5 patients (eGFR 8 ± 1 mL/min 1.73 m 2 ) scheduled for peritoneal dialysis catheter insertion and 15 controls (10M/5F, eGFR 99 ± 6 mL/min 1.73 m 2 ). Second, the effects of uraemic serum on the TLR4/NLRP3 inflammasome pathway were studied in C2C12 cells. In the muscle of CKD subjects, NLRP3 mRNA as well as its protein were overexpressed (by ~16-fold, respectively, P < 0.05 both vs. controls). Both IL-1β and IL-18 mRNA expressions were also up-regulated (~11.8–3.2-fold, respectively, P < 0.05). Also, cleaved caspase-1 was overexpressed in CKD muscle samples ( P < 0.001 vs. controls). Both muscle NLRP3 mRNA ( n = 22, r = −0.606, P < 0.01) and logIL-1 β protein ( n = 26, r = −0.460, P < 0.02) were inversely associated with residual renal function, which suggests that the inflammasome is progressively activated in skeletal muscle of CKD patients as the residual renal function deteriorates. In addition, we observed that in C2C12 myotubes, uraemic serum up-regulates NLRP3 mRNA (~11-fold increase, P < 0.05), cleaved caspase-1 (by ~5-fold, P < 0.05), Il-1β mRNA (~3-fold increase, P < 0.05) and oxidative stress markers respect to normal serum. These effects were prevented by TAK-242, a selective TLR4 inhibitor. Overall, our data demonstrate the activation of TLR4/NLRP3/caspase-1 inflammasome and its downward inflammatory cascade in the muscle of subjects with advanced-stage CKD and suggest targeting TLR4/NLRP3 inflammasome as a new therapeutic strategy to blunt muscle inflammation in CKD.
It is unknown to what degree of sarcopenia related to heart failure (HF) is reversible with resolution of the HF syndrome. We evaluated whether (1) weight loss prior to left ventricular assist device (LVAD) is associated with pre-operative sarcopenia as quantified on pre-operative chest CTs and (2) determine the relationship between weight recovery (increase) after LVAD implantation and reduction of NT-proBNP levels. In a large single-centre cohort ( n = 502), CT measures of sarcopenia (pectoralis muscle mass indexed to body surface area and tissue attenuation) were correlated with pre-LVAD BMI trend ( n = 190). BMI and NT-proBNP trends before and after LVAD implantation were evaluated ( n = 403). Linear effects modelling was performed to test the association between NT-proBNP and BMI trends. A downtrending BMI prior to LVAD was associated with pectoralis muscle tissue attenuation ( P < 0.05). BMI declined prior to LVAD, declined further early post-implant, and then increased between 100 and 300 days post-implant (average per cent change in BMI in Year 1, 7.6%, 95% CI: 6.3–8.8%). NT-proBNP decreased during the first 100 post LVAD days (−5.4%, 95% CI: −6.6 to −4.2%). Post-LVAD NT-proBNP and BMI trends were significantly associated, with a decrease of 1 unit log NT-proBNP associated with an increase in BMI of 0.81 kg/m 2 (CI: 0.53–1.09, P < .001). The rise in post-LVAD BMI occurred after the reduction in NT-proBNP levels. Patients who failed to gain weight post-LVAD had the highest 6-month post-LVAD natriuretic peptides (lowest per cent BMI gain tertile NT-proBNP: 2208 vs. highest 1635 pg/mL, P < 0.001). Weight recovery during LVAD support occurs after the reduction in natriuretic peptide levels. Failure to gain weight during LVAD support was associated with persistently elevated natriuretic peptide levels. These data collectively suggest that recovery of body mass may be dependent upon recovery of the HF syndrome.
Patients with pancreatic cancer often lose weight during chemotherapy with associated changes in body composition. The goal of the present analysis was to describe changes in body composition in pancreatic cancer patients on an exercise regimen. The long-term goal is to determine whether an exercise intervention may attenuate changes in body composition and function. Twenty-two pancreatic cancer patients of all stages who were to receive chemotherapy were recruited into a pre-post exercise intervention study. A standard exercise prescription was individualized to include aerobic, resistance, stretch, and balance exercises. Pre- and post-intervention computed tomography-derived measures of body composition [skeletal muscle index (SMI), skeletal muscle density, visceral fat area, and subcutaneous fat area] and physical function measures (grip strength, timed up and go, 30-s chair stand, and tandem balance stand) were evaluated using paired t -tests, χ 2 tests, and Pearson correlation coefficients. The subjects were, on average, 62 years of age, 55% were female, 95% non-Hispanic White, and 45% were Stage IV. Body composition changes included a median 4.6% decrease in SMI ( P = 0.04), 7.91% increase in skeletal muscle density ( P = 0.05), 25.07% decrease in visceral fat area ( P = 0.0001), and 22.08% decrease in subcutaneous fat area ( P = 0.001). Adherence to aerobic and strength exercise was 65% and 57%, respectively. Some physical function measures improved, though not significantly: chair stands increased from a mean of 11.5 to 13.0 ( P = 0.59) and timed up and go improved from a mean of 11.7 to 10.3 ( P = 0.26). Change in right hand grip strength was marginally positively associated with changes in SMI ( r = 0.53, P = 0.06). Improvements in skeletal muscle density were seen in 63% of patients, including Stage IV patients but did not correlate with change in function. Exercise is feasible during neoadjuvant chemotherapy for pancreatic cancer patients of all stages and may assist with maintaining physical function and improving body composition. Further research is needed.
Abstract Background An increase in waist circumference (WC) is a factor in lifestyle‐related diseases. The rectus abdominis muscle is a skeletal muscle that attaches to the pelvis from the xiphoid process and is thought to be affected by kyphosis deformity and posterior pelvic tilt. The purpose of this study is to examine differences between sacral‐abdominal wall distance (SAD) and WC and to determine whether they are associated with fall risk, frailty, markers of sarcopenia (grip strength and lean body mass), and spinal alignment. A secondary objective is to examine these differences by stratification by grip strength. Methods This retrospective study included 239 women aged 65 years or older (mean age 76.5 ± 6.7 years) attending an outpatient osteoporosis clinic. Bone mineral density and skeletal body composition (muscle mass index and trunk lean mass) were measured using dual‐energy X‐ray absorptiometry. SAD, pelvic tilt, and sagittal longitudinal axis were measured from simple X‐ray images of the spine sides. WC, grip strength, frailty, and fall risk score were investigated. Statistics were performed using Stat Flex, with two‐sided P < 0.05 being significantly different. Results WC was correlated with SAD (R = 0.68, P < 0.001). The SAD cut‐off value for a WC of 90 cm was 167 mm. The relationship between grip strength, SAD, and WC, weaker grip strength was associated with greater SAD; however, no significant difference was noted in WC. WC was not correlated with pelvic alignment but was correlated with body mass index (P < 0.01). Meanwhile, SAD was correlated with body mass index, pelvic tilt, sagittal longitudinal axis (P < 0.01), spinal alignment, and WC. Logistic regression analysis was performed with a grip strength of less than 18 kg as the objective variable. We found that the conditions for a grip strength of less than 18 kg were older age (P < 0.001), increased SAD (P = 0.02), and decreased trunk lean body mass. There was a decrease in grip strength (P < 0.05) and an increase in frailty (P < 0.05) and falls (P < 0.01) score in patients with SAD of 167 mm or greater. Conclusions SAD and WC were found to be correlated; SAD was associated with body weight, posterior pelvic tilt, and anterior spinal tilt deformity, while WC was related to body weight. Increased SAD was found to be linked with decreased grip strength and increased risk of falls. This study was the first to examine a new measurement, SAD, for its utility in assessing grip strength, spinal alignment, frailty, and fall risk.
Abstract Background Inflammation is a hallmark of cachexia; however, effective anti‐inflammatory treatments have not yet been identified. Interleukin‐1 receptor‐associated kinase 4 (IRAK4) is a key signalling node linking interleukin‐1 receptor (IL‐1R) and toll‐like receptor (TLR) activation to the production of multiple proinflammatory cytokines that are elevated in cancer cachexia. The purpose of this work is to evaluate whether pharmacological inhibition of IRAK4 kinase activity with PF‐06426779 could prevent cachexia using a model of pancreatic cancer. The effect of appetite stimulation via the ghrelin receptor agonist anamorelin was also examined as a benchmark of clinically validated mechanisms. Methods Female C57Bl/6J mice were given an intraperitoneal injection of KrasG12D; p53R172H; Pdx1‐Cre (KPC) pancreatic tumour cells. PF‐06426779 or anamorelin treatment was initiated at the onset of anorexia. Body weight and food intake were measured throughout the study. Body composition, muscle function (force), and physical activity (treadmill running endurance) were assessed at the end of the study. Results Chronic treatment with PF‐06426779, at doses covering in vitro IC50 and IC90 at Cmin, did not increase body weight, food intake, and muscle function in the KPC tumour model. In contrast, anamorelin (vs. vehicle) increased food intake (P < 0.01), hindlimb skeletal muscle mass (P < 0.01), and muscle strength (P < 0.05); however, treadmill running endurance was not increased. Conclusions These data suggest that inhibition of IRAK4 kinase activity is not sufficient to treat cachexia, at least in pancreatic cancer, and exploration of alternative anti‐inflammatory strategies that increase appetite is required.
Abstract Background Acute skeletal muscle wasting during critical illness is common and causes significant morbidity and functional limitation. Myofibre necrosis is a major histological finding but is often considered an unprogrammed by‐product of muscle inflammation. This study sought to evaluate if a form of programmed necrosis, necroptosis, is activated in skeletal muscle during critical illness. Methods A cohort of 28 patients from the MUSCLE‐UK study (ClinicalTrials.gov: NCT01106300) with serum and skeletal muscle biopsy samples were identified. Samples were available from ICU admission (T1) and between day 7–10 post admission (T2). Skeletal muscle was stratified by a histopathologist in the original study as necrotic (NEC, N = 14) or non‐necrotic (NONEC, N = 14) using haematoxylin and eosin staining. We used phosphorylated mixed‐lineage kinase domain‐like (pMLKL) protein (a key terminal effector protein) and receptor‐interacting protein kinase 3 (RIPK3) as markers of necroptosis activation using Western blotting and immunohistochemistry. Results We show that pMLKL expression is significantly higher in the NEC group [NEC: T2:T1 expression; 9.1 (IQR 3.9–22.3) vs. NONEC: T2:T1 expression; 0.9 (IQR 0.6–1.1), P = 0.003]. We then confirm this upregulation and describe co‐localization with receptor interacting protein kinase 3 (RIPK3) in skeletal muscle using immunohistochemistry. We show that both RIPK3 and pMLKL are present within intact myofibres at the intermediate timepoint day 3 without cellular infiltrate. At T2, pMLKL is also present in the interstitial space where there is infiltrate of CD68 positive immune cells. The observed necroptosis may originate from both internal and infiltrating sources. These findings were absent in samples from patients who did not exhibit histopathological features of necrosis. Conclusions We show that necroptosis machinery, RIPK3 and pMLKL, are associated with conventional histopathological features of myonecrosis in a critically ill cohort.
Abstract Introduction Critical illness associated with intensive care unit (ICU) admission often results in persistent skeletal muscle wasting and may lead to frailty in older and patients with multi‐morbidity. Early recognition of patients at high‐risk of long‐term complications could provide opportunities to minimize the impact of critical illness and improve health and quality of life. MicroRNAs (miRs) are short, non‐coding RNAs that regulate approximately two‐thirds of the human genome and are involved in most biological processes. Multiple studies have demonstrated their role in muscle development and disease and their potential as biomarkers of muscle wasting. Aim and methods This systematic review examined the potential of miRs as biomarkers and therapeutics for muscle wasting during and following critical illness. PubMed and Scopus databases were searched for terms associated with critical illness, ICU, muscle wasting, frailty and microRNAs from inception to June 2022 (PROSPERO number CRD42022339531). Results Out of 537 articles, seven studies met the inclusion criteria and examined skeletal muscle and circulating miRs in the context of muscle wasting and/or frailty related to critical illness. Across the seven studies, 27 different miRs were identified that were reported to be dysregulated in the muscle and four in the blood, plasma or serum of critically ill patients. Four miRs were reported to be altered in both muscle and blood during critical illness and their levels moderately correlated with parameters of muscle function. These included canonical muscle‐enriched miRs (myomiRs), such as miR‐133, miR‐1 and miR‐181, which correlated with muscle strength in critically ill patients. However, most of the miRs reported to be dysregulated in the muscle following critical illness were examined in one article only. Conclusions This systematic review highlights the potential of miRs as biomarkers of skeletal muscle wasting and ICU‐associated weakness following critical illness, suggesting the need for larger validation studies using unbiased techniques. We have described circulating and muscle microRNAs, which correlated with muscle parameters during critical illness. However, the limited number of studies in this area highlights the requirement for further studies before these could be considered in clinical practice.
Restrictions on outdoor movements due to the coronavirus disease (COVID-19) pandemic have led to a decreased physical activity; this can lead to sarcopenia and frailty in older adults. Our recent study has demonstrated a significant decrease in the trunk muscle mass immediately after the pandemic's first wave (April–May 2020) among Japanese community-dwelling older women. In the present study, we further examined whether muscle mass recovery or deterioration occurs after 1 year of the pandemic's first wave by comparing physical measurements among the following assessment periods: before the first wave, immediately after the first wave, and at 1-year follow-up thereafter. This study included 77 women (78.0 ± 5.7 years) who underwent physical measurements for muscle mass, grip strength, one-leg stand-up ability (3 s), and oral motor skills and answered questionnaires on sociality (social network, participation, and support) in the three assessment periods. The frequency of going out and the subjective vitality were significantly decreased immediately after the first wave; these recovered at the 1-year follow-up ( P < 0.001). When comparing muscular measures, the trunk muscle mass index preferentially decreased immediately after the first wave but recovered significantly at the 1-year follow-up ( P < 0.001). Conversely, the appendicular skeletal muscle mass index (ASMI) and grip strength continued to decrease until the 1-year follow-up ( P < 0.001 and P = 0.03, respectively). The ability to perform a one-leg stand-up for 3 s and the oral motor skills did not change significantly across the assessment periods. The prevalence of pre-sarcopenia and sarcopenia tended to increase during these periods ( P = 0.068). The reduction and subsequent recovery patterns for sociality were similar to those observed for the trunk muscle mass. Our findings demonstrated differences in the reversibility of skeletal muscle mass and strength at 1 year after the first wave of the COVID-19 pandemic: the trunk muscle mass declined acutely and recovered rapidly, whereas the ASMI and grip strength declined continuously. These differences in the skeletal muscle recovery and deterioration might help formulate short-term or long-term strategies for COVID-19-related sarcopenia prevention in community-dwelling older adults.
Abstract Background Loss of skeletal muscle mass is prevalent among patients affected by chronic kidney disease (CKD). It is associated with significant morbidity and mortality. The underlying molecular pathogenesis has yet to be fully understood. The aim of this systematic review is to summarize the current evidence on molecular changes in the skeletal muscle of humans and rodents with CKD and to assess the strength of such evidence. Methods The PubMed and EMBASE databases were searched using three main themes: messenger ribonucleic acid/protein/microRNA expression, skeletal muscle and CKD. This study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) standards. Results A total of 98 studies were included in the systematic review, comprising 26 prospective human clinical studies, four human and rodent studies, and 68 rodent‐only studies (32 mouse and 36 rat models respectively). The sample sizes of human studies were largely small (40% of studies had ≤20 participants). Qualitative polymerase chain reaction (qPCR) was the most commonly used method for gene expression and none of the studies fulfilled the Minimum Information for Publication of qPCR Experiments criteria for quality assessment. Majority of the studies investigated only a few genes or a specific signalling pathway. FBXO32, TRIM63, MSTN, IL6, TNF and IGF1 were the most investigated genes. The identified differentially expressed genes and proteins belonged to eight major pathways, including apoptosis, autophagy, inflammation, insulin/insulin‐like growth factor 1 signalling, lipid metabolism, mitochondrial function, muscle cell growth and differentiation, and protein degradation, similar to other chronic disease states. Conclusions The current evidence regarding molecular alterations in the skeletal muscle in CKD is largely derived from small and heterogenous studies. Markedly similar modifications in the major biological pathways between CKD and other chronic diseases supports shared deleterious molecular mechanisms producing muscle atrophy, irrespective of the underlying specific disease.
AbstractBackgroundPreoperative skeletal muscle deficiency is an established risk factor for poor survival outcomes in patients with renal cell carcinoma (RCC). However, given the dynamic nature of skeletal muscle associated with malignancy, there is a need to evaluate the prognostic benefit of muscle area change from the preoperative to postoperative period. We hypothesize that an improvement in muscle area following nephrectomy, measured by linear segmentation of L3 psoas and paraspinal musculature, is associated with improvement in overall survival (OS) and cancer specific survival (CSS) for patients with pT3 and pT4 RCC.MethodsWe retrospectively analysed 270 pT3 and pT4 RCC patients who underwent nephrectomy from March 2004 to February 2020 with available preoperative and postoperative axial CT or MRI studies segmented at the L3 vertebrae. The majority were N0 (79%) and M0 (68%). Psoas and paraspinal muscles were measured bilaterally using a validated digital ruler tool. Total muscle area (TMA) was calculated by aggregating the area of all four muscles and total muscle area index (TMI) by dividing the TMA by height squared (m2). The prognostic value of postoperative muscle improvement, defined as any increase in muscle area index, was analysed using Kaplan–Meier and Cox proportional stepwise hazard models.ResultsMedian time between preoperative scans and surgery was approximately 22 days and between surgery and postoperative scans 172 days. One hundred twenty‐one patients (44.8%) had an increase in total muscle area index post‐nephrectomy (IQR = 33.4; P ≤ 0.0001). On Kaplan–Meier analysis, postoperative improvement in TMI was associated with decreased odds of mortality (P = 0.0024) with a median follow‐up of 38.6 months. In a multivariable Cox regression analysis, improvement of TMI was associated with increased OS (HR = 0.52, 95% CI 0.35–0.78, P < 0.001) and increased CSS (HR = 0.55, 95% CI 0.32–0.94, P = 0.030). A 5% or more improvement in TMI was also associated with increased OS (HR = 0.53, 95% CI 0.34–0.84, P = 0.006) and increased CSS (HR = 0.46, 95% CI 0.24–0.86, P = 0.015).ConclusionsAny improvement in TMI between preoperative and initial postoperative imaging after nephrectomy was associated with increased OS and CSS in patients with pT3 and pT4 RCC. Perioperative linear segmentation is an efficient tool that may improve current prognostication methods and can be performed on any imaging software platform.
Abstract Background Chronic obstructive pulmonary disease (COPD) is associated with skeletal muscle mitochondrial dysfunction. Resistance exercise training (RT) is a training modality with a relatively small pulmonary demand that has been suggested to increase skeletal muscle oxidative enzyme activity in COPD. Whether a shift into a more oxidative profile following RT also translates into increased mitochondrial respiratory capacity in COPD is yet to be established. Methods This study investigated the effects of 13 weeks of RT on m. vastus lateralis mitochondrial capacity in 11 persons with moderate COPD [45% females, age: 69 ± 4 years (mean ± SD), predicted forced expiratory volume in 1 s (FEV1): 56 ± 7%] and 12 healthy controls (75% females, age: 66 ± 5 years, predicted FEV1: 110 ± 16%). RT was supervised and carried out two times per week. Leg exercises included leg press, knee extension, and knee flexion and were performed unilaterally with one leg conducting high‐load training (10 repetitions maximum, 10RM) and the other leg conducting low‐load training (30 repetitions maximum, 30RM). One‐legged muscle mass, maximal muscle strength, and endurance performance were determined prior to and after the RT period, together with mitochondrial respiratory capacity using high‐resolution respirometry and citrate synthase (CS) activity (a marker for mitochondrial volume density). Transcriptome analysis of genes associated with mitochondrial function was performed. Results Resistance exercise training led to similar improvements in one‐legged muscle mass, muscle strength, and endurance performance in COPD and healthy individuals. In COPD, mitochondrial fatty acid oxidation capacity and oxidative phosphorylation increased following RT (+13 ± 22%, P = 0.033 and +9 ± 23%, P = 0.035, respectively). Marked increases were also seen in COPD for mitochondrial volume density (CS activity, +39 ± 35%, P = 0.001), which increased more than mitochondrial respiration, leading to lowered intrinsic mitochondrial function (respiration/CS activity) for complex‐1‐supported respiration (−12 ± 43%, P = 0.033), oxidative phosphorylation (−10 ± 42%, P = 0.037), and electron transfer system capacity (−6 ± 52%, P = 0.027). No differences were observed between 10RM and 30RM RT, nor were there any adaptations in mitochondrial function following RT in healthy controls. RT led to differential expression of numerous genes related to mitochondrial function in both COPD and healthy controls, with no difference being observed between groups. Conclusions Thirteen weeks of RT resulted in augmented skeletal muscle mitochondrial respiratory capacity in COPD, accompanied by alterations in the transcriptome and driven by an increase in mitochondrial quantity rather than improved mitochondrial quality.
Abstract Background The age‐related loss of muscle mass and quality, sarcopenia, has many contributing factors, one of which may be cellular senescence, but this is not well defined in human skeletal muscle. Method Primary cells were isolated from biopsy samples of the vastus lateralis muscle from healthy adult males (n = 6, 22 ± 1 years), sorted (magnetic activated cell sorting) and chemically induced (doxorubicin, DOX, 0.2 μM) to a senescent state. This allowed the parallel and simultaneous investigation of the two main skeletal muscle‐derived cell types: satellite cell‐derived CD56+ve/desmin+ve myoblasts (muscle precursor cells) and CD56− ve/TE7+ve fibroblasts (at >95% purity). Both cell types were followed for up to 35 days post DOX treatment with a combination of quantitative immunocytochemistry and qRT‐PCR for senescent markers and senescence‐associated secretory phenotype (SASP) factors. Results Myoblasts and fibroblasts showed temporal and quantitative differences in many of the senescence markers studied. p16 protein expression increased across the time course (P < 0.0001) with no difference between cell types, whereas at the mRNA level, myoblasts showed increased p16 expression from 4 days post treatment (FC = 3.03 ± 0.99), and in fibroblasts, this appeared later at 10 and 35 days post DOX treatment (FC = 8.09 ± 2.46, P < 0.0001). Both myoblasts (FC = 8.83 ± 1.72) and fibroblasts (FC = 2.33 ± 1.10) showed significant increases in p21 mRNA (P < 0.0001), which remained elevated in the myoblast cell populations across the 35‐day time course but returned to baseline in the fibroblasts from 4 days post DOX treatment. Within 35 days post DOX treatment, all cell populations of both myoblasts and fibroblasts had reached 100% SA‐β‐Gal‐positive cells (P < 0.05). γH2aX expression (a marker of DNA damage) increased 1 day after DOX treatment in the myoblasts (FC = 3.3 ± 1.1, P < 0.05) but returned to baseline within 4 days post DOX treatment, whereas fibroblasts showed a similar trend that did not reach statistical significance. Significant reductions in expression of the proliferation marker Ki67 1 day post DOX treatment were seen in both cell types and were maintained throughout the time course (FC = 0.11 ± 0.07, P < 0.0001). Significant changes over the time course were also observed in mRNA expression of selected SASP factors (e.g. PAI‐1, MMP3, and IGFBP3, P < 0.05). Conclusions Neither cellular senescence nor sarcopenia is fully understood. The present data on human primary myoblasts and fibroblasts obtained from the same tissue sample show that senescence is a complex, non‐linear, and dynamic cellular process which shows intra‐ and inter‐cell variability.
The poor survival of pancreatic cancer patients is largely attributable to cachexia, a syndrome of severe weight and muscle loss. To investigate the aetiology of cancer cachexia, preclinical models that closely recapitulate the human disease process are essential. Patient derived tumour organoids are promising novel cancer models, but their ability to induce cachexia in mice has not been investigated. We developed two pancreatic tumour organoid-based mouse models and demonstrate their potential for cancer cachexia research. Two patients with pancreatic cancer, from whom tumour organoid cultures were previously established, were selected based on their cachexia phenotype. Patient 09 was characterized as cachectic according to the international consensus definition of cancer cachexia, whereas patient 12 was classified as non-cachectic. Organoid cultures PANCO-09b and PANCO-12a in basement membrane extract (BME) were injected subcutaneously into the flanks of 9-weeks old NMRI- Foxn1 nu mice ( n = 8/group). A control group was injected with BME only ( n = 4). Body weight was monitored every 2–3 days for 38 days. Hind limb muscle wet and dry weights were measured. Adipocyte size in inguinal white adipose tissue was measured using haematoxylin and eosin-stained sections. Expression of genes associated with cancer cachexia in muscle and liver tissue was analysed using qPCR. Engraftment of tumour organoids was successful in 87.5% of PANCO-09b implanted mice and in 50% of PANCO-12a mice, with similar average tumour weights at endpoint (34.4 ± 25.1 mg vs. 32.8 ± 40.2 mg, respectively, P = 0.450). All groups initially gained weight, but PANCO-12a implanted mice progressively lost an average body weight of 1.7 ± 0.8 g from day 28 onwards. PANCO-12a-implanted mice gained significantly less weight from baseline than controls (0.7 ± 0.6 g, P = 0.027). Overall body weight gain of PANCO-09b mice was also lower but not significantly different from controls (2.0 ± 1.2 g vs. 2.9 ± 1.6 g, P = 0.961). Wet weights of hind leg muscles were negatively correlated with tumour weight but did not differ between groups. Adipocytes of PANCO-12a implanted mice were smaller compared to SHAM as well as PANCO09b mice ( P < 0.0001), indicative of white adipose tissue wasting. Implantation of human pancreatic tumour organoids into mice negatively affects their body weight, but does not recapitulate body weight loss of donor patients. The reduced adipocyte size and inverse correlation between tumour weights and muscle weights in these mice are consistent with early-stage cachexia or pre-cachexia. This study shows that implantation of tumour organoids into mice provides a valuable model to investigate the processes underlying the heterogeneous presentation of cancer cachexia.
AbstractBackgroundLong‐term exposure to microgravity during spaceflight has adverse effects on human health including muscle atrophy, impaired immune function, and alterations in gut microbiome profile. Gut microorganisms influence a wide range of host biological processes, but their interactions with skeletal muscle and the immune system under microgravity have yet to be elucidated.MethodsRhesus macaques (Macaca mulatta) were subjected to −6° head‐down tilted bed rest (HDBR) for 6 weeks. Faecal samples, skeletal muscle tissue, and peripheral blood mononuclear cells (PBMCs) were collected for metagenomic, metabolomic, and transcriptomic analyses, respectively, and further integrated for a multi‐omics analysis.ResultsHead‐down tilted bed rest significantly altered taxon abundance (P < 0.05) in 1 class, 5 orders, 11 families, 55 genera, and 122 species of microbes. We also identified the significantly changed metabolites (P < 0.05, fold change >1.2, variable importance in projection >1) in atrophied muscles, including some crucial metabolites (such as l‐alanine and l‐carnitine) and hub metabolites (such as pyridoxamine and epinephrine) involved in energy metabolism. Transcriptomic analysis of PBMCs revealed genes related to leucocyte activation, differentiation, and interleukin‐2 production that were differentially expressed as a result of HDBR exposure (fold change >2 and P < 0.05). By integrating multi‐omics analysis, we identified three bacterial genera (Klebsiella, Kluyvera, and Bifidobacterium) that were closely associated with immune dysfunction and five (including Oligella, Sporosarcina, Citrobacter, Weissella, and Myroides) that were associated with abnormal metabolism of amino acids in atrophied muscles induced by HDBR. The reduced abundance of butyrate‐producing colon bacteria Eubacterium, Roseburia, and their cross‐feeding bacteria Bifidobacteria may contribute to the impaired immune function and muscle atrophy caused by HDBR.ConclusionsThis is the first report of the HDBR‐associated changes in gut microbiota composition, metabolomics of skeletal muscle, and transcripts of PBMCs in a non‐human primate. The underlying microbiota–muscle and microbiota–immune interactions during simulated microgravity imply that modulation of gut microbiota may represent a novel strategy for enhancing the health and safety of crew members during long‐term space expeditions.
AbstractBackgroundSkeletal muscle mass is regulated by intracellular anabolic and catabolic activities. Increased catabolic activity can shift the balance towards net protein breakdown and muscle atrophy. Mitochondrial oxidative stress activates catabolism and is linked to muscle loss. Reducing mitochondrial oxidative stress is thus a plausible approach to prevent muscle atrophy. We tested this concept in age‐dependent muscle atrophy by genetically overexpressing the mitochondrial antioxidant thioredoxin‐2 (TXN2).MethodsWe tested the functional role of TXN2 using ageing (n = 7–10 per group) and denervation (n = 3 per group) models in a transgenic mouse line that overexpresses TXN2. We investigated if overexpression of TXN2 blocks muscle loss in these models by examination of muscle weight, fibre size, and fibre number in young (~7 months) and aged (~26 months) TXN2‐transgenic mice and controls. We studied the underlying mechanisms by mRNA and protein assays including transcriptomic profiling, western blot analysis, immunostaining, as well as succinate dehydrogenase, dihydroethidium, and terminal deoxynucleotidyl transferase dUTP nick end labelling staining.ResultsOverexpression of TXN2 did not significantly alter the baseline skeletal muscle size, weight, fibre type distribution, or expression of mitochondrial respiratory chain components, but it did preserve muscle mass during ageing. The hindlimb muscle mass in aged TXN‐transgenic mice was ~21–24% greater (in tibialis anterior, gastrocnemius/soleus combined, and tibialis anterior/extensor digitorum longus combined) than in age‐matched controls (all P < 0.05). The reduction in both muscle fibre number (872 ± 206 vs, 637 ± 256 fibres in extensor digitorum longus muscle, P < 0.05) and muscle fibre size (1959 ± 296 vs. 1477 ± 564 μm2 in tibialis anterior muscle, P < 0.05) seen in young vs. aged control muscles was not significant in young vs. aged TXN‐transgenic mice (both P > 0.05). Transcriptomic analysis revealed that catabolic genes that are up‐regulated in ageing muscle, including those subserving apoptosis and the ubiquitin‐like conjugation system, were normalized by TXN2 overexpression. Further, overexpressing TXN2 suppressed oxidative stress and caspase‐9/3‐mediated apoptotic signalling in the aged muscle at the protein level. Although denervation and its effects have been considered a component of age‐related muscle atrophy, TXN2 overexpression failed to attenuate atrophy in an acute denervation model (TXN‐transgenic vs. control mice, P > 0.05), despite preventing denervation‐induced oxidative stress and apoptosis.ConclusionsMitochondrial oxidative stress appears to play a crucial role in effecting chronic age‐dependent, but not acute neurogenic, muscle atrophy. Increased TXN2 protects muscle against oxidative stress‐associated catabolic activity in ageing muscle and thus is a potential therapeutic approach to attenuate age‐related muscle atrophy.
Abstract Background Cachexia is a debilitating complication of cancer characterized by progressive wasting and weakness of skeletal muscles that reduces quality of life and can compromise survival. Many anticancer treatments, such as chemotherapy, also cause muscle wasting, which impairs the response to treatment. Given that many cancer patients present with cachexia at the initiation of treatment, we investigated whether cachectic mice were susceptible to chemotherapy‐induced muscle wasting and to investigate contributing mechanisms, including the dysregulation of microRNAs (miRs). Methods Cachectic colon‐26 (C‐26) tumour‐bearing mice were given 5‐fluourouracil (5‐FU) chemotherapy or vehicle treatment and analysed for muscle mass, fibre size and composition, and miR expression. Mechanisms were validated in vitro using C2C12 cell culture and miR mimics and inhibitors and were confirmed in vivo by injecting muscles of 5‐FU‐treated cachectic mice with recombinant adeno‐associated viral (rAAV) vectors encoding a miR sponge. Results In cachectic tumour‐bearing mice, 5‐FU chemotherapy exacerbated the loss of skeletal muscle mass compared with vehicle treatment (by −12% to −20%, P < 0.05). miR expression profiling, quantitative real‐time PCR, and in vitro analyses revealed contributing mechanisms including miR‐351‐3p‐dependent ERK2 inhibition. Intramuscular injection of rAAV vectors encoding a sponge to reduce miR‐351‐3p expression in 5‐FU‐treated cachectic mice enhanced ERK phosphorylation (+18%, P < 0.05) and increased muscle fibre size (+15%, P < 0.01). Hsa‐miR‐125a‐3p shares similar predicted gene targets as mmu‐miR‐351‐3p, and its inhibition in human muscle cells in vitro prevented 5‐FU‐induced atrophy (P < 0.001) and increased ERK phosphorylation (P < 0.001). Conclusions The findings implicate miR‐351‐3p‐mediated ERK2 inhibition as a contributing mechanism in chemotherapy‐induced muscle wasting in mice with cancer cachexia and that its inhibition is a promising adjunct therapy for preserving muscles during cancer treatment.
AbstractBackgroundPhysical activity is associated with a lower risk of colorectal cancer (CRC) and CRC‐specific mortality. However, evidence for a causal relationship between physical activity and disease progression is lacking. Here, we have used CRC organoids to create a novel mouse model for spontaneous metastasis formation to multiple organs. We have used this model to assess the influence of voluntary exercise on disease progression.MethodsCollagen‐embedded murine colorectal tumour organoids were transplanted into the livers of immunocompetent C57Bl/6 mice using microsurgery. Voluntary exercise in tumour‐bearing mice was modelled by offering running wheels continuously (n = 12) or 3 h/day (n = 12) versus no wheel access (n = 12). Running wheel revolutions were cumulatively measured every 30 min and physical activity was continuously monitored by infrared cameras. Food intake was monitored throughout the experiment and body composition was assessed with echoMRI. Animals were sacrificed 14 weeks after tumour initiation. Tumour load was quantified by EpCAM immunohistochemistry staining. Systemic inflammation parameters were assessed in blood plasma by a multiplex immunoassay.ResultsTumour growth was initiated by implantation of CRC organoids into the livers of immunocompetent mice. The resulting tumours spontaneously formed distant metastases to non‐implanted liver lobes and to the lungs. Mice with access to the running wheels for 3 h/day ran relatively short distances (2.3 ± 0.3 km/night; 221 ± 29 km total distance) with relatively high intensity (wheel revolutions/h). Mice with continuous access to the running wheels ran significantly longer distances (6.6 ± 3.0 km/night; 600 ± 290 km total distance) with a significantly lower intensity. Both exercise groups showed increased lean body mass, and decreased fat mass and body weight compared with tumour‐bearing control mice. Food intake was unaffected by exercise or tumour growth. Primary tumour growth was not significantly affected by exercise. However, mice with continuous wheel access (long distance‐lower intensity group) displayed increased lung metastasis and decreased liver metastasis formation, when compared with the sedentary control group. Short distance‐higher intensity exercise did not affect metastasis formation. Analysis of blood cytokine levels revealed that mice with continuous wheel access displayed signs of systemic inflammation.ConclusionsThese results suggest that exercise has the potential to influence the patterns and extent of metastasis in CRC, and that the degree and intensity of exercise are likely to be important variables. Confirmation of these results in additional preclinical models with or without systemic treatment is warranted.
Abstract Background Pre‐frailty has been identified as a clinically silent mechanism pre‐disposing people to frailty. The goal of this study is to investigate the impact of low‐dose metformin on pre‐diabetic pre‐frail patients (>50 years) on skeletal muscle mass, speed of gait, handgrip power, and health‐related quality of life (HR‐QoL). Methods We did a retrospective cohort study of subjects aged 50 years and older who were diagnosed with pre‐frailty (one or two criteria present based on Fried Frailty Index) and pre‐diabetes (HbA1c 5.7% to 6.4%) from May 2018 to April 2020. Subjects taking low‐dose metformin were compared with non‐metformin participants through a review of the medical records. The results were analysed at baseline and 6–12 months post‐prescription with or without metformin. All data were accompanied by a 95% confidence interval. Results One hundred and thirteen pre‐diabetic and pre‐frail subjects were recruited to metformin [the mean metformin administration was 750 (140) mg/day] (n = 58) or control group (n = 55). The average age was 66.3 (6.8) years old, with 52.2% of the population being female. The baseline demographic, nutritional, physical, and mental status data did not differ between groups. In comparison with the control group, an 8.9 (1.8) month low‐dose metformin intervention resulted in a higher skeletal muscle mass index of 1.26 (1.66) kg/m2 (P = 0.029), a faster gait speed of 0.15 (0.22) m/s (P = 0.011), and a greater handgrip strength of 2.1 (2.9) kg (P = 0.037). However, there was no difference in myostatin serum levels or HR‐QoL between groups. Conclusions Low‐dose metformin was statistically and clinically meaningful to improve the original skeletal muscle mass index, gait speed, and grip strength as part of the sarcopenia dimension, but the Euro Quality of Life‐5 Dimensions index and myostatin serum levels did not change significantly.
