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Background: The p97 complex participates in the degradation of muscle proteins during atrophy upon fasting or denervation interacting with different protein adaptors. We investigated whether and how it might also be involved in muscle wasting in cancer, where loss of appetite occurs, or amyotrophic lateral sclerosis (ALS), where motoneuron death causes muscle denervation and fatal paralysis., Methods: As cancer cachexia models, we used mice bearing colon adenocarcinoma C26, human renal carcinoma RXF393, or Lewis lung carcinoma, with breast cancer 4T1-injected mice as controls. As ALS models, we employed 129/SvHsd mice carrying the mutation G93A in human SOD1. The expression of p97 and its adaptors was analysed in their muscles by quantitative real-time polymerase chain reaction (qPCR) and western blot. We electroporated plasmids into muscles or treated mice with disulfiram (DSF) to test the effects of inhibiting p97 and nuclear protein localization protein 4 (Nploc4), one of its adaptors, on atrophy., Results: The mRNA levels of p97 were induced by 1.5-fold to 2-fold in tibialis anterior (TA) of all the cachectic models but not in the non-cachectic 4T1 tumour-bearing mice (P ≤ 0.05). Similarly, p97 was high both in mRNA and protein in TA from 17-week-old SOD1 G93A mice (P ≤ 0.01). Electroporation of a shRNA for murine p97 into mouse muscle reduced the fibre atrophy caused by C26 (P = 0.0003) or ALS (P ≤ 0.01). When we interrogated a microarray, we had previously generated for the expression of p97 adaptors, we found Derl1, Herpud1, Nploc4, Rnf31, and Hsp90ab1 induced in cachectic TA from C26-mice (Fold change > 1.2, adjusted P ≤ 0.05). By qPCR, we validated their inductions in TA of cachectic and ALS models and selected Nploc4 as the one also induced at the protein level by 1.5-fold (P ≤ 0.01). Electroporation of a CRISPR/Cas9 vector against Nploc4 into muscle reduced the fibre atrophy caused by C26 (P = 0.01) or ALS (P ≤ 0.0001). Because DSF uncouples p97 from Nploc4, we treated atrophying myotubes with DSF, and found accumulated mono and polyubiquitinated proteins and reduced degradation of long-lived proteins by 35% (P ≤ 0.0001), including actin (P ≤ 0.05). DSF halves Nploc4 in the soluble muscle fraction (P ≤ 0.001) and given to C26-bearing mice limited the body and muscle weight loss (P ≤ 0.05), with no effect on tumour growth., Conclusions: Overall, cancer cachexia and ALS seem to display similar mechanisms of muscle wasting at least at the catabolic level. The p97-Nploc4 complex appears to have a crucial role in muscle atrophy during these disorders and disrupting this complex might serve as a novel drug strategy., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Cancer cachexia (CC) is a debilitating multifactorial syndrome, involving progressive deterioration and functional impairment of skeletal muscles. It affects about 80% of patients with advanced cancer and causes premature death. No causal therapy is available against CC. In the last few decades, our understanding of the mechanisms contributing to muscle wasting during cancer has markedly increased. Both inflammation and oxidative stress (OS) alter anabolic and catabolic signaling pathways mostly culminating with muscle depletion. Several preclinical studies have emphasized the beneficial roles of several classes of nutraceuticals and modes of physical exercise, but their efficacy in CC patients remains scant. The route of nutraceutical administration is critical to increase its bioavailability and achieve the desired anti-cachexia effects. Accumulating evidence suggests that a single therapy may not be enough, and a bimodal intervention (nutraceuticals plus exercise) may be a more effective treatment for CC. This review focuses on the current state of the field on the role of inflammation and OS in the pathogenesis of muscle atrophy during CC, and how nutraceuticals and physical activity may act synergistically to limit muscle wasting and dysfunction.

Background: Skeletal muscle is characterized by an efficient regeneration potential that is often impaired during myopathies. Understanding the molecular players involved in muscle homeostasis and regeneration could help to find new therapies against muscle degenerative disorders. Previous studies revealed that the Ser/Thr kinase p21 protein-activated kinase 1 (Pak1) was specifically down-regulated in the atrophying gastrocnemius of Yoshida hepatoma-bearing rats. In this study, we evaluated the role of group I Paks during cancer-related atrophy and muscle regeneration., Methods: We examined Pak1 expression levels in the mouse Tibialis Anterior muscles during cancer cachexia induced by grafting colon adenocarcinoma C26 cells and in vitro by dexamethasone treatment. We investigated whether the overexpression of Pak1 counteracts muscle wasting in C26-bearing mice and in vitro also during interleukin-6 (IL6)-induced or dexamethasone-induced C2C12 atrophy. Moreover, we analysed the involvement of group I Paks on myogenic differentiation in vivo and in vitro using the group I chemical inhibitor IPA-3., Results: We found that Pak1 expression levels are reduced during cancer-induced cachexia in the Tibialis Anterior muscles of colon adenocarcinoma C26-bearing mice and in vitro during dexamethasone-induced myotube atrophy. Electroporation of muscles of C26-bearing mice with plasmids directing the synthesis of PAK1 preserves fiber size in cachectic muscles by restraining the expression of atrogin-1 and MuRF1 and possibly by inducing myogenin expression. Consistently, the overexpression of PAK1 reduces the dexamethasone-induced expression of MuRF1 in myotubes and increases the phospho-FOXO3/FOXO3 ratio. Interestingly, the ectopic expression of PAK1 counteracts atrophy in vitro by restraining the IL6-Stat3 signalling pathway measured in luciferase-based assays and by reducing rates of protein degradation in atrophying myotubes exposed to IL6. On the other hand, we observed that the inhibition of group I Paks has no effect on myotube atrophy in vitro and is associated with impaired muscle regeneration in vivo and in vitro. In fact, we found that mice treated with the group I inhibitor IPA-3 display a delayed recovery from cardiotoxin-induced muscle injury. This is consistent with in vitro experiments showing that IPA-3 impairs myogenin expression and myotube formation in vessel-associated myogenic progenitors, C2C12 myoblasts, and satellite cells. Finally, we observed that IPA-3 reduces p38α/β phosphorylation that is required to proceed through various stages of satellite cells differentiation: activation, asymmetric division, and ultimately myotube formation., Conclusions: Our data provide novel evidence that is consistent with group I Paks playing a central role in the regulation of muscle homeostasis, atrophy and myogenesis., (© 2018 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.)

Muscle wasting occurs during various chronic diseases and precedes death in humans as in mice. The evaluation of the degree of muscle atrophy in diseased mouse models is often overlooked since it requires the sacrifice of the animals for muscle examination or expensive instrumentation and highly qualified personnel, such as Magnetic Resonance Imaging (MRI). Very often behavioral tests for muscle strength evaluation are used as an outcome measurement in preclinical therapeutic trials. However, these tests are easy to perform serially, but not enough sensitive to detect early muscle changes during disease progression. Monitoring muscle loss in living animals could allow to perform more informative preclinical trials with a better evaluation of therapeutic benefit with respect to muscle wasting. We developed a non-invasive procedure based on micro-computed tomography (micro-CT) without contrast agents to monitor hind limb muscle wasting in mouse models of amyotrophic lateral sclerosis (ALS) and cancer cachexia: the transgenic SOD1G93A mouse and the colon adenocarcinoma C26-bearing mouse, respectively. We established the scanning procedure and the parameters to consider in the reconstructed images to calculate the Index of Muscle Mass (IMM). The coefficient of variance for the whole procedure was 2.2%. We performed longitudinally micro-CT scan of hind limbs in SOD1G93A mice at presymptomatic and symptomatic stages of the disease and calculated the IMM. We found that IMM in SOD1G93A mice was lower than age-matched controls even before symptom onset. We also detected a further decrease in IMM as disease progresses, most markedly just before disease onset. We performed the same analyses in the C26-based mouse model losing quickly body and muscle mass because of cancer cachexia. Overall, we found that the reduced muscle content detected by micro-CT mirrored the reduced muscle weight in both disease models. We developed a fast, precise and easy-to-conduct imaging procedure to monitor hind limb muscle mass, useful in therapeutic preclinical trials but also in proof-of-principle studies to identify the onset of muscle wasting. This method could be widely applied to other disease models characterized by muscle wasting, to assist drug development and search for early biomarkers of muscle atrophy. Moreover, reducing the number of mice needed for the experiments and being less distressing are in line with the 3R principle embodied in national and international directives for animal research., Competing Interests: The authors have declared that no competing interests exist.

Cancer cachexia is a life-threatening syndrome that affects most patients with advanced cancers and causes severe body weight loss, with rapid depletion of skeletal muscle. No treatment is available. We analyzed microarray data sets to identify a subset of genes whose expression is specifically altered in cachectic muscles of Yoshida hepatoma-bearing rodents but not in those with diabetes, disuse, uremia or fasting. Ingenuity Pathways Analysis indicated that three genes belonging to the C-X-C motif chemokine receptor 4 (CXCR4) pathway were downregulated only in muscles atrophying because of cancer: stromal cell-derived factor 1 (SDF1), adenylate cyclase 7 (ADCY7), and p21 protein-activated kinase 1 (PAK1). Notably, we found that, in the Rectus Abdominis muscle of cancer patients, the expression of SDF1 and CXCR4 was inversely correlated with that of two ubiquitin ligases induced in muscle wasting, atrogin-1 and MuRF1, suggesting a possible clinical relevance of this pathway. The expression of all main SDF1 isoforms (α, β, γ) also declined in Tibialis Anterior muscle from cachectic mice bearing murine colon adenocarcinoma or human renal cancer and drugs with anticachexia properties restored their expression. Overexpressing genes of this pathway (that is, SDF1 or CXCR4) in cachectic muscles increased the fiber area by 20%, protecting them from wasting. Similarly, atrophying myotubes treated with either SDF1α or SDF1β had greater total protein content, resulting from reduced degradation of overall long-lived proteins. However, inhibiting CXCR4 signaling with the antagonist AMD3100 did not affect protein homeostasis in atrophying myotubes, whereas normal myotubes treated with AMD3100 showed time- and dose-dependent reductions in diameter, until a plateau, and lower total protein content. This further confirms the involvement of a saturable pathway (that is, CXCR4). Overall, these findings support the idea that activating the CXCR4 pathway in muscle suppresses the deleterious wasting associated with cancer.

Background: The loss of skeletal muscle mass (atrophy) that accompanies disuse and systemic diseases is highly debilitating. Although the pathogenesis of this condition has been primarily studied in mammals, Drosophila is emerging as an attractive system to investigate some of the mechanisms involved in muscle growth and atrophy., Results: In this review, we highlight the outstanding unsolved questions that may benefit from a combination of studies in both flies and mammals. In particular, we discuss how different environmental stimuli and signaling pathways influence muscle mass and strength and how a variety of disease states can cause muscle wasting., Conclusions: Studies in Drosophila and mammals should help identify molecular targets for the treatment of muscle wasting in humans., (Copyright © 2013 Wiley Periodicals, Inc.)

The p97/VCP ATPase complex facilitates the extraction and degradation of ubiquitinated proteins from larger structures. We therefore studied if p97 participates to the rapid degradation of myofibrillar proteins during muscle atrophy. Electroporation of a dominant negative p97 (DNp97), but not the WT, into mouse muscle reduced fibre atrophy caused by denervation and food deprivation. DNp97 (acting as a substrate-trap) became associated with specific myofibrillar proteins and its cofactors, Ufd1 and p47, and caused accumulation of ubiquitinated components of thin and thick filaments, which suggests a role for p97 in extracting ubiquitinated proteins from myofibrils. DNp97 expression in myotubes reduced overall proteolysis by proteasomes and lysosomes and blocked the accelerated proteolysis induced by FoxO3, which is essential for atrophy. Expression of p97, Ufd1 and p47 increases following denervation, at times when myofibrils are rapidly degraded. Surprisingly, p97 inhibition, though toxic to most cells, caused rapid growth of myotubes (without enhancing protein synthesis) and hypertrophy of adult muscles. Thus, p97 restrains post-natal muscle growth, and during atrophy, is essential for the accelerated degradation of most muscle proteins.