Andrea David Re Cecconi

Muscle atrophy occurs during physiological (i.e., fasting, aging) and pathological (i.e., amyotrophic lateral sclerosis, cancer) conditions and anticipates death. Since not all patients will undergo muscle wasting, it would be highly useful to identify them soon to intervene early. We have studied the promoters/enhancers of a subset of atrophy-related genes or atrogenes upregulated in muscles of rodents during various kinds of atrophy (i.e., disuse, diabetes, cancer, fasting, uremia) for their ability to induce early atrophy. Comparing their upstream non-coding regions, using as backbone MuRF1 promoter (one of the earliest muscle-specific genes induced by wasting), we cloned various promoters upstream of a doubled reporter system (FLuciferase/tdTomato). Through in vitro and in vivo studies in mice subjected to denervation or cancer injection, we selected a sequence (i.e., MyoRep) able to predict atrophy upon cut of the sciatic nerve or cancer. In vivo imaging of MyoRep mice emit a bioluminescent signal earlier than muscle loss. Importantly, MyoRep was unable to sense atrophy during fasting or physiological variations following the circadian rhythms. Since MyoRep can discriminate muscle loss due to pathological conditions from physiological ones anticipating wasting, it represents an unprecedented tool to predict it early in various diseases with local or systemic atrophy.

Intensive efforts have been made to identify features that could serve as biomarkers of aging. Yet, drug-based interventions aimed at lessening the detrimental effects of getting older are lacking. This is largely attributable to tissue-specificity, sex-related differences, and to the difficulty of identifying actionable targets, which continues to pose a significant challenge. Here, we implement a bioinformatics approach revealing that aging-associated increase of the transmembrane Ectodysplasin-A2-Receptor is a prominent tissue-independent alteration occurring in humans and other species, and is particularly pronounced in models of accelerated aging. We show that strengthening of the Ectodysplasin-A2-Receptor signalling axis in myogenic precursors and differentiated myotubes suffices to trigger potent parainflammatory responses, mirroring aspects of aging-driven sarcopenia. Intriguingly, obesity, insulin-resistance, and aging-related comorbidities, such as type-2-diabetes, result in heightened levels of the Ectodysplasin-A2 ligand. Our findings suggest that targeting the Ectodysplasin-A2 surface receptor represents a promising pharmacological strategy to mitigate the development of aging-associated phenotypes.

Cachexia is a metabolic syndrome consisting of massive loss of muscle mass and function that has a severe impact on the quality of life and survival of cancer patients. Up to 20% of lung cancer patients and up to 80% of pancreatic cancer patients are diagnosed with cachexia, leading to death in 20% of them. The main drivers of cachexia are cytokines such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), macrophage inhibitory cytokine 1 (MIC-1/GDF15) and transforming growth factor-beta (TGF-β). Besides its double-edged role as a tumor suppressor and activator, TGF-β causes muscle loss through myostatin-based signaling, involved in the reduction in protein synthesis and enhanced protein degradation. Additionally, TGF-β induces inhibin and activin, causing weight loss and muscle depletion, while MIC-1/GDF15, a member of the TGF-β superfamily, leads to anorexia and so, indirectly, to muscle wasting, acting on the hypothalamus center. Against this background, the blockade of TGF-β is tested as a potential mechanism to revert cachexia, and antibodies against TGF-β reduced weight and muscle loss in murine models of pancreatic cancer. This article reviews the role of the TGF-β pathway and to a minor extent of other molecules including microRNA in cancer onset and progression with a special focus on their involvement in cachexia, to enlighten whether TGF-β and such other players could be potential targets for therapy.

Cancer cachexia consists of dramatic body weight loss with rapid muscle depletion due to imbalanced protein homeostasis. We found that the mRNA levels of apelin decrease in muscles from cachectic hepatoma-bearing rats and three mouse models of cachexia. Furthermore, apelin expression inversely correlates with MuRF1 in muscle biopsies from cancer patients. To shed light on the possible role of apelin in cachexia in vivo, we generated apelin 13 carrying all the last 13 amino acids of apelin in D isomers, ultimately extending plasma stability. Notably, apelin D-peptides alter cAMP-based signaling in vitro as the L-peptides, supporting receptor binding. In vitro apelin 13 protects myotube diameter from dexamethasone-induced atrophy, restrains rates of degradation of long-lived proteins and MuRF1 expression, but fails to protect mice from atrophy. D-apelin 13 given intraperitoneally for 13 days in colon adenocarcinoma C26-bearing mice does not reduce catabolic pathways in muscles, as it does in vitro. Puzzlingly, the levels of circulating apelin seemingly deriving from cachexia-inducing tumors, increase in murine plasma during cachexia. Muscle electroporation of a plasmid expressing its receptor APJ, unlike apelin, preserves myofiber area from C26-induced atrophy, supporting apelin resistance in vivo. Altogether, we believe that during cachexia apelin resistance occurs, contributing to muscle wasting and nullifying any possible peptide-based treatment.

Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N and 520fs) induce myofibril abnormalities, altered nuclear morphology and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of βPS integrin and Sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant, aberrantly accumulate α- and β-Dystroglycan and α-Sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration.

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.

Trabectedin (ET743) and lurbinectedin (PM01183) limit the production of inflammatory cytokines that are elevated during cancer cachexia. Mice carrying C26 colon adenocarcinoma display cachexia (i.e., premature death and body wasting with muscle, fat and cardiac tissue depletion), high levels of inflammatory cytokines and subsequent splenomegaly. We tested whether such drugs protected these mice from cachexia. Ten-week-old mice were inoculated with C26 cells and three days later randomized to receive intravenously vehicle or 0.05 mg/kg ET743 or 0.07 mg/kg PM01183, three times a week for three weeks. ET743 or PM01183 extended the lifespan of C26-mice by 30% or 85%, respectively, without affecting tumor growth or food intake. Within 13 days from C26 implant, both drugs did not protect fat, muscle and heart from cachexia. Since PM01183 extended the animal survival more than ET743, we analyzed PM01183 further. In tibialis anterior of C26-mice, but not in atrophying myotubes, PM01183 restrained the NF-κB/PAX7/myogenin axis, possibly reducing the pro-inflammatory milieu, and failed to limit the C/EBPβ/atrogin-1 axis. Inflammation-mediated splenomegaly of C26-mice was inhibited by PM01183 for as long as the treatment lasted, without reducing IL-6, M-CSF or IL-1β in plasma. ET743 and PM01183 extend the survival of C26-bearing mice unchanging tumor growth or cachexia but possibly restrain muscle-related inflammation and C26-induced splenomegaly.

Physical activity improves the prognosis of cancer patients, partly by contrasting the associated muscle wasting (cachexia), through still unknown mechanisms. We asked whether aerobic exercise causes secretion by skeletal muscles of proteins (myokines) that may contrast cachexia. Media conditioned by peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α)-expressing myotubes, reproducing some metabolic adaptations of aerobic exercise, as increased mitochondrial biogenesis and oxidative phosphorylation, restrained constitutively active Forkhead box-containing subfamily O3 (caFoxO3)-induced proteolysis. Microarray analysis identified amphiregulin (AREG), natriuretic peptide precursor B (NppB), musclin and fibroblast growth factor 18 (FGF18) as myokines highly induced by PGC1α. Notably, only musclin tended to be low in muscle of mice with a rare human renal carcinoma; it was reduced in plasma and in muscles of C26-bearing mice and in atrophying myotubes, where PGC1α expression is impaired. Therefore, we electroporated the Tibialis Anterior (TA) of C26-bearing mice with musclin or (its receptor) natriuretic peptide receptor 3 (Npr3)-encoding plasmids and found a preserved fiber area, as a result of restrained proteolysis. Musclin knockout (KO) mice lose more muscle tissue during growth of two distinct cachexia-causing tumors. Running protected C26-bearing mice from cachexia, not changing tumor growth, and rescued the C26-induced downregulation of musclin in muscles and plasma. Musclin expression did not change in overloaded plantaris of mice, recapitulating partially muscle adaptations to anaerobic exercise. Musclin might, therefore, be beneficial to cancer patients who cannot exercise and are at risk of cachexia and may help to explain how aerobic exercise alleviates cancer-induced muscle wasting.

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 andin vitroby dexamethasone treatment. We investigated whether the overexpression of Pak1 counteracts muscle wasting in C26‐bearing mice andin vitroalso during interleukin‐6 (IL6)‐induced or dexamethasone‐induced C2C12 atrophy. Moreover, we analysed the involvement of group I Paks on myogenic differentiationin vivoandin vitrousing 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 andin vitroduring 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 ofatrogin‐1andMuRF1and possibly by inducingmyogeninexpression. Consistently, the overexpression of PAK1 reduces the dexamethasone‐induced expression ofMuRF1in myotubes and increases the phospho‐FOXO3/FOXO3 ratio. Interestingly, the ectopic expression of PAK1 counteracts atrophyin vitroby 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 atrophyin vitroand is associated with impaired muscle regenerationin vivoandin 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 within vitroexperiments 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.