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.

Cancer cachexia associates with a systemic autophagy-inducing activity mimicked by cancer cell-derived IL-6 trans-signaling.

The majority of cancer patients with advanced disease experience weight loss, including loss of lean body mass. Severe weight loss is characteristic for cancer cachexia, a condition that significantly impairs functional status and survival. The underlying causes of cachexia are incompletely understood, and currently no therapeutic approach can completely reverse the condition. Autophagy coordinates lysosomal destruction of cytosolic constituents and is systemically induced by starvation. We hypothesized that starvation-mimicking signaling compounds secreted from tumor cells may cause a systemic acceleration of autophagy during cachexia. We found that IL-6 secreted by tumor cells accelerates autophagy in myotubes when complexed with soluble IL-6 receptor (trans-signaling). In lung cancer patients, were cachexia is prevalent, there was a significant correlation between elevated IL-6 expression in the tumor and poor prognosis of the patients. We found evidence for an autophagy-inducing bioactivity in serum from cancer patients and that this is clearly associated with weight loss. Importantly, the autophagy-inducing bioactivity was reduced by interference with IL-6 trans-signaling. Together, our findings suggest that IL-6 trans-signaling may be targeted in cancer cachexia.

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.

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.

Clinical classification of cancer cachexia: phenotypic correlates in human skeletal muscle.

BACKGROUND: Cachexia affects the majority of patients with advanced cancer and is associated with a reduction in treatment tolerance, response to therapy, and duration of survival. One impediment towards the effective treatment of cachexia is a validated classification system. METHODS: 41 patients with resectable upper gastrointestinal (GI) or pancreatic cancer underwent characterisation for cachexia based on weight-loss (WL) and/or low muscularity (LM). Four diagnostic criteria were used >5%WL, >10%WL, LM, and LM+>2%WL. All patients underwent biopsy of the rectus muscle. Analysis included immunohistochemistry for fibre size and type, protein and nucleic acid concentration, Western blots for markers of autophagy, SMAD signalling, and inflammation. FINDINGS: Compared with non-cachectic cancer patients, patients with LM or LM+>2%WL, mean muscle fibre diameter was reduced by about 25% (p = 0.02 and p = 0.001 respectively). No significant difference in fibre diameter was observed if patients had WL alone. Regardless of classification, there was no difference in fibre number or proportion of fibre type across all myosin heavy chain isoforms. Mean muscle protein content was reduced and the ratio of RNA/DNA decreased in patients with either >5%WL or LM+>2%WL. Compared with non-cachectic patients, SMAD3 protein levels were increased in patients with >5%WL (p = 0.022) and with >10%WL, beclin (p = 0.05) and ATG5 (p = 0.01) protein levels were increased. There were no differences in phospho-NFkB or phospho-STAT3 levels across any of the groups. CONCLUSION: Muscle fibre size, biochemical composition and pathway phenotype can vary according to whether the diagnostic criteria for cachexia are based on weight loss alone, a measure of low muscularity alone or a combination of the two. For intervention trials where the primary end-point is a change in muscle mass or function, use of combined diagnostic criteria may allow identification of a more homogeneous patient cohort, reduce the sample size required and enhance the time scale within which trials can be conducted.

Biosynthesis of riboflavin. Screening for an improved GTP cyclohydrolase II mutant.

GTP cyclohydrolase II catalyzes the first dedicated step in the biosynthesis of riboflavin and appears to be a limiting factor for the production of the vitamin by recombinant Bacillus subtilis overproducer strains. Using error-prone PCR amplification, we generated a library of the B. subtilis ribA gene selectively mutated in the GTP cyclohydrolase II domain. The ratio of the GTP cyclohydrolase II to 3,4-dihydroxy-2-butanone synthase activities of the mutant proteins was measured. A mutant designated Construct E, carrying seven point mutations, showed a two-fold increase in GTP cyclohydrolase II activity and a four-fold increase in the K(m) value with GTP as the substrate. Using the analog 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate as the substrate, the mutant showed a rate enhancement by a factor of about two and an increase in the K(m) value by a factor of about 5. A series of UV absorption spectra obtained in stopped-flow experiments using the wild-type and mutant enzymes revealed isosbestic points indicative of apparently perfect reactions, which were similar to the findings obtained with GTP cyclohydrolase II of Escherichia coli. Initial burst velocities obtained for the mutant and wild-type proteins were similar. The data suggest that the mutations present in Construct E are jointly conducive to the acceleration of a late step in the reaction trajectory, most probably the release of product from the enzyme.