European Journal of Translational Myology
The European Journal of Translational Myology (previously published as Basic and Applied Myology) is recognized as the authoritative European forum in the field of Translational Myology. The journal provides a comprehensive platform for the dissemination of high-quality research addressing muscle biology and pathology across multiple levels of investigation, including fundamental, cellular, molecular, genetic, clinical, and translational studies. Its scope encompasses a broad array of article types, such as original research papers, systematic and narrative reviews, editorials, viewpoints, methodological protocols, rapid communications, comments, and letters to the editor. Through this multifaceted approach, the journal fosters scholarly exchange and advances the integration of basic and applied knowledge into clinical and translational practice.
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34 | Hyaluronan enhances proliferation and myogenic differentiation of C2C12 murine myoblasts under inflammatory stress
Hyaluronan (HA) is a non-sulfated glycosaminoglycan, widely used for medical and pharmaceutical applications including tissue muscle repair. A recent study demonstrates that HA activates muscle stem cells to repair damaged muscle. When muscle damage occurs, stem cells start producing and coating themselves with hyaluronic acid, driven by demethylase JMJD3, which allows muscle stem cell adaptation to inflammation and the initiation of muscle repair (Nakka et al., 2022). The aim of this work is to characterize the effect of Multifractionated - HA on myoblast rescue under inflammatory conditions, using C2C12 murine muscle cell proliferation and differentiation. In this study, we investigated the potential of a HA mixture (2-1000 KDa, 1 mg/mL, Regenflex TeM, Regenyal Laboratories SRL) in increasing the proliferative and myogenic capacity of myoblasts in the presence or absence of pro-inflammatory agents (IL-1β, LPS), known to impair proliferation. Results revealed that TeM significantly improved reparative mechanisms and exerts a strong pro-proliferative effect, enhancing wound healing within 24 hours post-injury, even in the presence of inflammatory conditions. Additionally, we evaluated the myogenic potential of C2C12 cells treated with TeM by analyzing the gene and protein expression of key myogenic markers, including IGF-1, MyoD, Myogenin, Mrf4, myogenin, and MHC-1, even under inflammatory conditions. Moreover, TeM treatment upregulated myogenic biomarkers, suggesting a positive impact on differentiation pathways. Regenflex T&M has been shown to increase the repair process in injured myoblast monolayers, to restore healing capacity under otherwise limiting conditions and to stimulate the muscle differentation process. This data streghtens the rationale for using infiltrative intramuscular T&M to promote the repair of injured muscles in clinical settings.
Abstracts of the 22nd Meeting of the Interuniversity Institute of Myology
Copyright (c) 2026 The Author(s) https://creativecommons.org/licenses/by-nc/4.0
43 | Synergistic negative effects of advanced glycation end products and tumor-conditioned medium on C2C12 myotubes and the protective activity of Vaccinium macrocarpon
Cancer cachexia (CC) is a complex and multifactorial syndrome characterized by progressive loss of skeletal muscle mass that could be worsened by unhealthy dietary habits, such as the consumption of a Western diet (WD), which supplies high quantities of advanced glycation end products (AGEs), non-enzymatic adducts formed through the reaction of reducing sugars with cellular macromolecules that can can alter the structure and function of the latter. An excessive accumulation of AGEs has been observed in both the plasma and tumor microenvironment of cancer patients, where it correlates with the severity of muscle wasting. In recent years, natural compounds have emerged as promising candidates for treatment due to their diverse bioactive properties. Among these, Vaccinium macrocarpon (VM) has been identified through preliminary ad hoc screening as a potent inhibitor of AGE formation and activity, suggesting its potential as a therapeutic agent against AGE-induced muscle wasting. Since the degeneration of the neuromuscular junction (NMJ) may contribute to CC-related muscle wasting, we explored the role of AGEs in the dismantling on NMJ and the therapeutic potential effect of VM, with a particular focus the effects on AChR formation and clustering, a well-established in vitro model for evaluating NMJ development and function. Our data indicate that AGEs cause the worsening of atrophy of C2C12 myotubes and a reduction of AChR clustering (an in vitro model of NMJ formation) induced by the treatment of cells with Lewis lung carcinoma conditioned medium (LLC-CM). Co-treatment with VM rescued myotube diameter and AChR clustering, suggesting its potential to mitigate the detrimental effects on skeletal muscle of WD associated with CC.
28 | Impairment of skeletal muscle force and mitochondrial respiratory capacity precedes muscle mass loss in an aged murine model of diet-induced obesity
Sarcopenic obesity (SO) is characterized by the coexistence of obesity and sarcopenia, conditions that synergistically exacerbate frailty, particularly in the elderly population. However, the mechanisms underlying the development of sarcopenia within the context of obesity remain poorly understood. It is well established that mitochondrial respiratory capacity in skeletal muscle declines with age, and it is plausible that this decline contributes to the pathogenesis of SO. In this study, using a diet-induced murine model of obesity, we investigated the progression of sarcopenia in obesity during aging, with a particular focus on skeletal muscle mitochondria. We analyzed 12-month-old female C57BL/6 mice fed either a high-fat diet (HFD; 60% of calories from fat) or a standard diet (SD; 10% of calories from fat) over a seven-month period. We monitored body weight and composition in vivo, muscle volume, muscle strength and endurance. At 1, 3, and 7 months of diet, muscles were collected from both HFD- and SD-fed mice for histological and molecular ex vivo analyses. We evaluated respiration, number, area, diameter of mitochindria and mitochondrial network. From the first month of HFD feeding, mice exhibited increased body weight and fat accumulation. Grip strength, used as an indicator of muscle function, was significantly reduced in HFD-fed mice after one month, with further decline observed at three months. By seven months, histological analysis revealed a marked reduction in myofiber cross-sectional area compared to SD-fed controls. Notably, mitochondrial respiratory capacity and mitochondrial number were initially elevated after one month of HFD but showed a significant decline by three months. These results suggest that early metabolic alterations in mitochondrial function contribute to the onset and progression of sarcopenia in the context of obesity.
45 | Interleukin-4 prevents cancer-induced muscle wasting and promotes exercise-like metabolic adaptations
Cancer cachexia is a complex syndrome characterized by body weight loss, muscle wasting, mitochondrial abnormalities and energy metabolism alterations. Previous findings showed that treating tumor-bearing (TB) mice with interleukin-4 (IL4) improved muscle fitness and myogenesis. IL4 and IL4 receptor were also found to be upregulated in the skeletal muscle after exercise. The present study aimed to investigate whether IL4 administration improves skeletal muscle energy metabolism in cancer hosts. Ten-weeks-old Balb/c male mice were inoculated with 5x105 C26 colon carcinoma cells and sacrificed at days 10 or 15. Daily IL4 treatment (66.5 μg/kg) was performed by intraperitoneal injection. The gastrocnemius muscle was used to assess mitochondrial proteins (western blot) and respiration (high-resolution respirometry, Oroboros Instruments), and to perform metabolomic and lipidomic profiling (HPLC-coupled mass spectrometry). Treatment with IL4 prevented loss of body weight, muscle mass and muscle strength in day 15 TB mice. The protein levels of OXPHOS complexes II and III, cytochrome c and PGC-1α in the skeletal muscle were significantly increased in treated vs untreated TB mice. Similar trends were found for TOMM20, while trends to decrease were found for BNIP3. OXPHOS complex II activity was significantly increased in treated vs untreated TB mice. Tumor- and IL4-driven modulations in skeletal muscle metabolomic profile were observed, involving many energy-associated metabolites. Similarly, the skeletal muscle lipidome was altered by tumor burden and was partially restored by IL4. IL4 treatment improves energy metabolism in the skeletal muscle of TB mice. This effect relies on the improvement of mitochondrial homeostasis and the increased availability and capacity of mitochondrial OXPHOS complexes, suggesting that IL4 may act as an exercise mimetic. Further efforts to unravel the mechanism underlying IL4 efficacy are required.
26 | Validation of a new multi-target antioxidant treatment for the therapy of Duchenne muscular dystrophy
Currently, there is no general cure for Duchenne muscular dystrophy (DMD); standard therapy with corticosteroids only slows the progression of the disease, while often causing significant side effects. Therefore, there is a clear need for new, mutation-independent effective therapeutics with favourable side effect profiles. In DMD, dystrophin deficiency triggers a series of pathophysiological events, e.g., calcium dysregulation, mitochondrial disfunction, oxidative stress, inflammatory and immune responses, and impaired muscle regeneration. Experimental therapies in the past usually focused on only one of these aspects at the time, limiting the efficacy of these approaches. A therapeutic strategy capable of simultaneously acting on multiple DMD pathophysiological features would therefore be highly desirable. To this aim, we identified the key transcriptional regulator NRF2 as a promising pharmacological target, which can be specifically activated by the small molecule Nacetylcysteine ethyl ester (NACET). Here we report the initial results obtained by treating young mdx mice with NACET from 6 to 10 weeks of age. So far, we found that the highest dosage led to a significant improvement of normalized force in hindlimb muscles and to a decrease in the percentage of necrotic fibers, in the absence of any adverse side effects.
16 | Immunoproteasome inhibition positively impacts the gut-muscle axis in Duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD), a fatal muscle-wasting disorder resulting from mutations in the DMDgene, is often compounded by secondary non-muscle complications. A key factor in DMD progression is chronic inflammation, triggered by the release of Danger Associated Molecular Patterns (DAMPs) following myofiber necrosis, which attracts proinflammatory cells to dystrophic muscle. Notably, gastrointestinal (GI) dysfunction, common in DMD patients, can also contribute to systemic inflammation via the gut microbiota. The immunoproteasome (IP) plays a crucial role in regulating antigen presentation, cytokine production, and immune cell responses, and acts as a first line of defense in the gut by modulating the NF-κB pathway. Our prior research demonstrated that IP inhibition alleviates molecular, histological, and functional deficits in mdx mice, a well-established DMD model. Here, we investigated the impact of IP inhibition on the gut-muscle axis in these mice. Treatment with the IP inhibitor ONX-0914 increased the presence of anti-inflammatory CD206+ M2 macrophages in both the colon and muscle of mdx mice, while partially reduced pro-inflammatory CD68+ M1 cells. This treatment also improved muscular mitochondrial activity, enhanced muscle performance, and decreased serum creatine phosphokinase (CPK) levels and fibrosis. Utilizing microbiota perturbation models, including broad-spectrum antibiotics and fecal microbiota transplantation from IP-inhibited mdx mice, we demonstrated that ONX-0914's beneficial effects on muscle function are mediated through microbiotadependent mechanisms. Our findings contribute to understanding the role of IP inhibition as a potential therapeutic strategy for modulating the dystrophic gut-muscle axis, suggesting new avenues for microbiota-targeted therapies.
37 | Characterization of novel cancer cachexia preclinical models implementing chemotherapy and surgery
Cancer cachexia (CC) is a multifactorial syndrome characterized by weight loss, metabolic alterations, and muscle wasting. Although the C26-mouse model is widely used to study CC, its rapid tumor growth and lack of metastasis fail to recapitulate cancer patient’s trajectory. In colorectal cancer, standard treatment includes chemotherapy and resection of the primary tumor, often followed by tumor relapse and/or metastasis. This study aims to characterize novel C26-based experimental protocols to investigate tissue-specific metabolic changes. Three treatment protocols were evaluated in 3-month-old female BALB/c mice subcutaneously inoculated with 5×10⁵ colon carcinoma cells (C26). Each protocol combined FOLFOX chemotherapy with surgical resection: (1) two doses pre-resection (C26+2F), (2) two doses pre- and post-resection (C26+4F), and (3) a single dose pre-resection (C26+F). Tumors were surgically excised 4 days post-chemotherapy. A group of control mice was included in each experiment. At sacrifice, muscles and tissues were collected for biochemical, histological, and metabolomic analyses. Tumor-bearing mice showed signs of cachexia in all experiments, with increasing severity according to chemotherapy duration. Few mice showed no evidence of primary tumor relapse, fully recovering their body weight and muscle mass. Others progressed to lung metastases, indicating that curative interventions lead to distinct trajectories even in syngeneic mice. Metabolomics analysis in gastrocnemius and liver revealed distinct metabolic profiles of control and tumor-bearing mice, with the no relapsing mice clustering closer to the controls. Muscle and liver exhibit distinct metabolic alterations, showing signs of potential recovery depending on treatment strategy and early intervention. Further investigations are required to identify affected metabolic pathways associated with either bad prognosis or cancer survivorship and recovery.
01 | Investigating skeletal muscle-endothelial cell crosstalk in plasticity and disease
The maintenance of skeletal muscle mass and function is a major clinical challenge in aging, cancer, muscular dystrophies, and related conditions. While much attention over the past two decades has focused on intracellular pathways like Akt-mTORC1, the roles of muscle perfusion and paracrine signaling between muscle fibers and endothelial cells (ECs) remain poorly understood. Emerging evidence suggests a bidirectional crosstalk between these cell types. However, in vivo tools to study this interaction with cell-type specificity and temporal control are still limited. To address this, we developed an EC-specific MetRS mouse model expressing a mutant methionyl-tRNA synthetase (MetRS^L274G) under the tamoxifen-inducible Cdh5-CreERT2 promoter. This allows temporally controlled, EC-specific incorporation of the methionine analog azidonorleucine (ANL) into nascent proteins in vivo. Administered via drinking water, ANL selectively labels EC proteins within defined windows. Labeled proteins can be visualized and quantified using click chemistry–based FUNCAT or enriched by immunoprecipitation for downstream mass spectrometry–based proteomic analysis. Combined with tissue clearing and light sheet microscopy, this enables 3D mapping of endothelial protein synthesis across skeletal muscle. To validate the model, we induced angiogenesis via unilateral AAV-VEGF overexpression (2×10¹⁰ U/muscle) in the gastrocnemius muscle, using the contralateral limb as control. Our preliminary results demonstrate successful and specific endothelial protein labeling with clear spatial distribution across muscle tissue. Interestingly, VEGF-induced angiogenesis correlates with increased muscle mass, fiber size, and protein synthesis. We are now using the model to explore how controlled muscle growth or atrophy influences ECs, and how primary EC changes affect muscle physiology. This work aims to provide new insights into muscle–vascular communication relevant to muscle health and disease.
32 | Trifolium pratense extract mitigates skeletal muscle atrophy: a nutraceutical approach for the prevention of sarcopenia
Sarcopenia, the age-related loss of muscle mass and function, increases the risk of frailty, fractures, and loss of independence, ultimately reducing quality of life and contributing to higher mortality. With life expectancy rising in Western countries, sarcopenia represents a growing yet unresolved public health challenge, as no pharmacological treatments are currently available. Recent interest in medicinal plants as potential therapeutic agents has highlighted their ability to counteract various pathological conditions, including muscle atrophy. This study explores the protective effects of native Mediterranean on skeletal muscle cells exposed to atrophic stimuli that mimic sarcopenia and disuse (e.g., dexamethasone, starvation). Additionally, we assess whether controlled cultivation protocols can influence the bioactive metabolite content of these plants, potentially enhancing their anti-sarcopenic properties. Among the tested species, hydroalcoholic extract of Trifolium pratense demonstrates promising anti-atrophic effects, effectively preserving C2C12 myotube diameter against TNF- α/IFN-γ-induced muscle wasting. However, its protective efficacy varies depending on cultivation conditions, with some enhancing its bioactivity while others showing no significant effect. Furthermore, Trifolium pratense completely abolishes TNF-α/IFN-γ-induced reactive oxygen species production, underscoring its antioxidant properties. These findings suggest that Trifolium pratense could serve as a promising nutraceutical intervention to counteract muscle atrophy associated with aging. Moreover, optimizing its cultivation conditions may enhance its therapeutic potential, paving the way for targeted agronomic strategies to maximize its bioactivity. By integrating plant-based interventions into sarcopenia management, this study contributes to the development of accessible, nonpharmacological strategies aimed at improving muscle health and quality of life in aging populations.
12 | In vitro study emulating muscle response to stimuli exploiting 3D tissue model
Understanding muscle responses to external stimuli is critical for advancing therapeutic strategies and vaccine delivery, yet human-based models remain limited. To emulate muscle responses to immunological and delivery-related stimuli in vitro, we developed a human 3D muscle-like tissue model coupled with peripheral blood mononuclear cells (PBMCs). Upon exposure to immunostimulatory agents, the system supported the recruitment and modulation of antigen-presenting cells, including monocytes and macrophages, while stromal elements promoted dendritic cell expansion and activation. PBMCs exhibited an early innate immune signature (encompassing IL-15, IL-6, TNF-α, IFN-γ, IP-10, MCP-1, and IFN-inducible genes) reflecting immune activation profiles relevant for protective responses. Muscle-resident cells fostered an immunostimulatory environment, while the stromal compartment enhanced IFN-related mediator expression, demonstrating the platform’s capability to investigate muscle-immune system interactions under controlled conditions. To further explore muscle responses to delivery stimuli, we assessed the internalization and modulatory effects of solid lipid nanoparticles (SLN) and poly(lactic-co-glycolic acid) (PLGA) nanoparticles within the 3D muscle constructs. Both nanoparticle types were progressively internalized by myofibers, exhibiting distinct localization and accumulation near nuclei. Additionally, we tested the capability of SLN and PLGA nanoparticles to modulate inflammatory cytokines (IL-1β, IL-6, TNF-α) under inflammatory stimulation, indicating their potential for controlled delivery and local immune modulation within muscle tissue. In summary, this human 3D muscle-based in vitro model effectively emulates muscle responses to immunological and delivery stimuli, offering a versatile platform to study muscle immunobiology and evaluate advanced delivery strategies, while reducing the reliance on animal models in preclinical research.
03 | The muscle out of breath? Limitations and adaptations in chronic diseases
Skeletal muscle demonstrates remarkable plasticity in response to physiological stimuli such as exercise and hypoxia. Chronic diseases like chronic obstructive pulmonary disease (COPD), chronic heart failure (CHF), and obstructive sleep apnea syndrome (OSAS) result in chronic or intermittent hypoxia, impairing oxygen delivery to muscle tissue. Sickle cell disease (SCD), a genetic hemoglobinopathy, represents a unique model characterized by a complex hypoxic environnement (chronic, acute, and intermittent) stemming from severe chronic hemolytic anemia and recurrent vaso-occlusive crises. These hypoxic conditions induce profound alterations in skeletal muscle, mostly including fiber-type switching, atrophy, reduced capillarization, and impaired oxidative metabolism. The extent of these changes appears to depend on the nature and severity of hypoxia, as well as the degree of physical inactivity. In COPD and CHF, exercise training is well established to mitigate muscle dysfunction. Conversely, in SCD, physical activity was long considered risky due to fears of provoking vaso-occlusive events. However, our research group conducted a randomized controlled trial demonstrating that moderate-intensity endurance training is not only safe in individuals with SCD, but also enhances physical capacity and attenuates muscle alterations. These findings pave the way for novel therapeutic strategies based on exercise to improve muscle structure and quality of life in this population. In contrast, the effects of exercise training on skeletal muscle structure in patients with OSAS remain unexplored, highlighting a critical gap in the current understanding of muscle adaptation in hypoxia-related disorders.
40 | Role of glucocorticoids in cancer cachexia: focus on lipocalin 2 and iron homeostasis
Cachexia is a severe wasting syndrome characterized by profound skeletal muscle atrophy that significantly contributes to mortality across various diseases, including cancer. However, its underlying mechanisms remain poorly understood. We previously identified major alterations of iron metabolism in the skeletal muscle of cachectic tumor-bearing mice and in particular a decrease in mitochondrial iron content, resulting in mitochondrial dysfunction. Lipocalin 2 (LCN2), an iron-chelating protein, has been identified as one of the most upregulated genes in various models of skeletal muscle atrophy. Being known the role of glucocorticoids in muscle wasting and that LCN2 is a transcriptional target of the glucocorticoid receptor (GR), we hypothesized its involvement in the dysregulation of iron metabolism during muscle wasting. Firstly, we confirmed the increased expression of LCN2 in the skeletal muscle of both C26 tumor-bearing mice and dexamethasone-treated mice, recapitulating the cachectic phenotype. Consistently, in vitro LCN2 overexpression induced a significant reduction in C2C12 myotube diameter and, oppositely, its silencing was sufficient to prevent dexamethasone-induced atrophy. Moreover, iron supplementation prevented glucocorticoid-induced atrophy in vitro and mitigated the cachectic phenotype in our two mouse models. Importantly, local expression of LCN2 promoted markers of iron deprivation in vivo. These results suggest that iron metabolism acts as a key contributor to skeletal muscle atrophy. Our findings shed light on both cancer and glucocorticoid-induced skeletal muscle wasting mechanisms, highlighting LCN2 as a potential therapeutic target for muscle wasting diseases.
Welcome to the 22nd Meeting of the Interuniversity Institute of Myology!
The 22nd Meeting of the Interuniversity Institute of Myology (IIM) took place in Assisi, Italy, from September 11 to 14, 2025, welcoming 106 participants from academia, industry, and patient organizations across Europe and the USA. The meeting provided an interdisciplinary forum for discussing muscle tissue from every perspective, combining scientific sessions with informal social events to foster networking. Early-career scientists played a central role, representing half of the attendees. Their involvement was supported by the High Training Course “Advanced Myology Update 2025,” jointly organized by the University of Perugia and IIM, which hosted 15 participants in lectures and roundtable discussions led by invited experts. The scientific program comprised six sessions, including 28 oral presentations and 36 posters. Four keynote lectures by Karyn Esser, Markus Rüegg, Giulio Cossu, and Roberta Sartori offered in-depth insights into muscle biology, neuromuscular disorders, and translational research. In line with IIM’s support for young scientists, presenters under 35 were eligible for Best Talk, Best Poster Blitz, and Best Poster awards. Winners were invited to join the IIM Young Committee, contributing to future meeting organization. Overall, the 22nd IIM Meeting confirmed its role as an inclusive and dynamic forum for the myology community. It successfully combined high-level science with opportunities for mentorship and collaboration, reinforcing IIM’s international relevance. We look forward to welcoming both long-standing members and new participants at the 23rd IIM Meeting in Assisi, September 9-12, 2026, for another engaging event full of science and shared enthusiasm.
Copyright (c) 2026 the Author(s) http://creativecommons.org/licenses/by-nc/4.0
27 | Polymeric nanoparticles as a favorable delivery system to enhance the anti-inflammatory action of n-palmitoylethanolamide, an endocannabinoid-like molecule
Chronic low-grade inflammation is linked to age-related muscle loss (sarcopenia), highlighting the potential of daily anti-inflammatory strategies. A growing body of evidence suggests that N-palmitoylethanolamide (PEA), a natural endocannabinoid-like compound with anti-inflammatory properties, could represent a safe therapeutic option to counteract sarcopenia. However, the low solubility of PEA significantly limits its systemic bioavailability. To overcome this limitation, our work was aimed to develop a nanoparticle-based delivery system intended to enhance its systemic availability. We compared two nanoparticle formulations: solid lipid nanoparticles (SLNs) and poly-(lactic-co-glycolic acid) (PLGA) polymeric nanoparticles. These delivery systems were evaluated in terms of cellular uptake, cytotoxicity, and impact on myogenic differentiation. Our results indicate that PLGA nanoparticles exhibit superior cellular internalization compared to SLNs, display lower cytotoxicity, and do not substantially interfere with the myogenic differentiation process in cell culture. Furthermore, PEA encapsulated within PLGA nanoparticles effectively activated its canonical target, peroxisome proliferator-activated receptor alpha (PPAR-α), and protected muscle cells from lipopolysaccharide (LPS)-induced cytotoxicity in vitro. These findings represent a critical step toward the preclinical evaluation of PEA as a potential therapeutic agent for counteracting sarcopenia, using murine models of age-related muscle wasting. Funding: European Union Next Generation EU” n. P2022LSW98 (PRIN-PNRR 2022 from Italian "Ministero dell'Università e della Ricerca".
35 | A novel 3D skeletal muscle model to study cancer cachexia
Cancer cachexia is a multisystem syndrome affecting cancer patients, characterized by progressive body weight loss, muscle wasting, increased mortality and poor quality of life. Skeletal muscle represents a main target of cancer cachexia, where tumor-derived inflammatory factors including IL-1, IL-6 and TNF-α trigger muscle degeneration through the activation of specific proteolytic pathways, ultimately causing a dramatic loss of muscle mass and function. Although several mouse models have been used to study cancer cachexia, the close interaction between tumor and muscle cells has been investigated primarily in 2D in vitro co-culture models. This approach revealed important molecular and metabolic dysfunctions of muscle cells when co-cultured with tumor cells. However, the use of 2D systems to study skeletal muscle differentiation/degeneration dynamics presents many limitations, including the lack of the characteristic spatial orientation of the myotubes. Here, we present a novel hydrogel-based 3D in vitro muscle model to study cancer cachexia. We generated bio-artificial muscle and extended the model by incorporating a co-culture with colorectal carcinoma cells, enabling direct cell–cell contact between cancer cells and myogenic cells. After differentiation in co-cultures with carcinoma cells, myoblasts showed a severe impairment of their myogenic potential. This model also allows to uncouple direct cellcell contacts while studying the effect of secreted factors by an indirect co-culture system. In addition, we found that the typical myofiber alignment observed in native muscle tissue is preserved in our constructs also in the presence of carcinoma cells. The ability to study cancer-related myogenic dysfunction in a soft 3D environment represents a novel tool to study cancer cachexia mechanisms and drug development, further reducing the need for animal experiments.
29 | RNA-based combinatorial multi-target therapy for the treatment of Duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD) is a severe and fatal X-linked desease, caracterize by the lack of dystrophin leading to structural fragility of muscle fibers and promotes a pro-catabolic metabolic imbalance. To date, no curative treatment is available, partly due to the difficulty in restoring full dystrophin expression and developing non-immunogenic approaches. Reactivation of utrophin, a dystrophin’s paralogous protein naturally downregulated by microRNAs, has shown strong potential to compensate for dystrophin deficiency. The RNA_DMD project aims to address three major challenges that are 1) to reactivate utrophin expression to compensate for the loss of dystrophin, 2) to inhibit pro-catabolic pathways to promote muscle growth, 3) to develop non-immunogenic vectors for effective and tolerable combination therapy. We hypothesize that combination therapy could both restore muscle structural integrity and prevent or reverse muscle wasting and associated loss of function. To study aim 2, we used the bicistronic plasmid expressing green fluorescent protein (GFP) and bearing our shRNA against FOXO1/3 or MURF1caring by an adeno-associated virus with a muscle specific serotype (AAV-MYO). To date, we test the efficiency of our AAV-MYO-shRNA in wild-type (WT) mice by electroporation in tibialis anterior (TA) (1010 vg/muslce) and systemically (1012 vg/mouse). And then, we moved to 3-week-old D2-mdx mice (a severe model of DMD). Our preliminary data highlights in WT animals, that combined electroporation of AAV-MYOshMuRF1 and AAV-MYO-shFOXO1/3 in the tibialis anterior muscle leads to a reduction in the expression of MuRF1 (-24%; P=0.04), FOXO1 (-36%; P=0.002), and 3 (-39%; P=0.03) genes, associated with muscle hypertrophy described by a +30% increase in CSA (P=0.03) of TA. In 3-week-old D2-mdx mice separate intramuscular injection of AAV-MYO-shMuRF1 and AAVMYO- shFOXO1/3 led to a reduction after 4 weeks of MuRF1 transcript (-45%; P<0.001) and FOXO1 respectively (-27%; P=0.04).
05 | Stimulation of muscle stem cells using side-streams from animal and plant production
Current estimates predict that global food production needs to increase and adapt in response to population growth and dietary changes. Better utilization of animal and plantbased side-streams from food production will be necessary to achieve a circular economy while maintaining the nutritional needs of the global population. Muscle health and regenerative properties are important during aging, diabetes, and normal skeletal muscle homeostasis, and are highly affected by nutrient intake. Muscle stem cells, also known as satellite cells, play a key role in muscle repair and regeneration. Metabolic pathways involving glucose utilization are essential for the proper functioning of these stem cells. Muscle stem cells and their regenerative properties have been shown to be stimulated by small peptides and chemical compounds like phytochemicals, influencing proliferation, glucose uptake, and protein synthesis. In this study, we explore the potential of hydrolysates from side-streams of the chicken industry and phytochemical-rich extracts from plant sidestreams in glucose uptake and muscle regeneration. Our data showed that peptides generated from chicken hydrolysates and extracts from cauliflower, kale, and apple stimulated the proliferation of bovine muscle cells using the Incucyte live cell system. Further analysis revealed that chicken hydrolysate increased glucose uptake and protein synthesis in both bovine and rat muscle cells through regulation of the MEK and ERK signaling pathways. In conclusion, we observe bioactive compounds in rest-raw materials from both plants and chicken that can be promising nutraceutical supplements for muscle health.
44 | Targeted delivery of mir-486 to skeletal muscle: efficacy, tropism, and integrin dependency of MYOAAV2A vs. AAV9
Adeno-Associated Viruses (AAVs) are emerging as a promising tool for gene therapy as a way to modulate gene expression in specific tissues. Of particular interest to us is the application of AAV-based approach for treating congenital myopathies. The use of AAV in the muscle is complicated by the abundance and large distribution of this tissue in the body, requiring high dosages of the gene-delivery vector with higher risks of side effects, mostly related to liver toxicity. To address this challenge, we designed and selected a strong expression cassette for miR-486, known to ameliorate myopathic symptoms, through in vitro screening, and loaded it in the recently developed myotropic AAV virus MyoAAV2A to enhance skeletal muscle transduction and targeting. This delivery system achieves a high transduction and expression of miR-486 in mouse striated muscle while de-targeting the liver and the lungs. The higher muscle trophism and efficiency of MYOAAV2A compared to its parental serotype AAV9 depend on the presence of the integrin recognition motif, RGD, in the hypervariable region VIII of the virus capsid, possibly making its infection efficiency dependent on the presence of RGD-binding integrins (particularly αVβ6) in the sarcolemma. Our current understanding of how αVβ6 expression varies with age, muscle type, and in non-muscle organs is still limited, yet this knowledge could be valuable for designing MYOAAV-based therapeutic strategies. Here, we analysed the correlation between the transduction efficiency of MyoAAV2A and αVβ6 expression across various muscles, organs and mouse ages, comparing it with AAV9 as an integrin-independent control serotype.
46 | Investigating the impact of the tumor secretome on in vitro human skeletal and cardiac muscle models of cancer-induced cachexia
Cancer cachexia is a complex and poorly diagnosed metabolic syndrome primarily characterized by depletion of skeletal muscle and adipose tissue. Although cachexia impacts multiple organs, including the heart, research has remained focused on skeletal muscle, with no robust in vitro model of cardiac cachexia available. Furthermore, current knowledge regarding cancer cachexia is mainly derived from animal studies, which lack direct translatability to human physiology. Therefore, this project aims to bridge these gaps by integrating an established murine model of cancer cachexia with advanced human in vitro systems and, ultimately, patient-derived tissue explants to investigate the effects of the tumor secretome on skeletal and cardiac muscles. Specifically, we developed fully human in vitro models by indirectly exposing immortalized myoblasts to the secretome of human colon carcinoma cells (HCT116) using transwell-based co-cultures, which led to reduced expression of mature myogenic markers and formation of smaller myotubes. Applying the same approach to human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, we observed impaired cardiac differentiation and maturation as well as reduced cardiomyocyte contractile function. Experiments with hiPSC-derived cardiac organoids, which offer increased complexity and greater physiological relevance, are ongoing. In parallel, since we have observed more small extracellular vesicles (sEVs) containing stress-related proteins in cachectic cancer patients compared to weight stable patients, we have begun testing the sEV inhibitor Manumycin A in our cell systems. These experiments showed reduced sEV secretion, and we plan to assess whether sEV inhibition can mitigate cachectic phenotypes in our skeletal and cardiac models. By leveraging state-of-the-art human models and sEV-modulating interventions, this research will provide crucial insights into cachexia-induced alterations in skeletal and cardiac muscles.
42 | RAGE-ablated skeletal muscles resist cancer-induced atrophy through Akt-GSK-3β-PGC-1α pathway and fast-to-slow myofiber transition
Cancer cachexia (CC) syndrome affects most patients with advanced cancer and is characterized by loss of body weight and muscle atrophy, leading to progressive functional impairment and reduced survival. The receptor for advanced glycation end-products (RAGE) is a multiligand receptor expressed during muscle development but repressed in adult healthy muscles. RAGE is re-expressed at myofiber level in tumor-bearing mice [2], but the specific contribution of muscular RAGE to CC was unknown. Using an HSA/Cre-Lox tamoxifen-inducible conditional AgermKO mouse model, in which RAGE is selectively ablated in myofibers, we demonstrated that, compared with control (Agerflox) mice, AgermKO mice showed reduced body and muscle weight loss, no significant reduction of myofiber cross-sectional areas and muscle functionality, restrained muscle and systemic inflammation, and increased survival, 25 days after s.c. injection of Lewis lung carcinoma (LLC) cells. Mechanistically, AgermKO muscles resist cancer-induced atrophy by maintaining an active Akt-GSK-3β-PGC-1α pathway and promoting a fast-to-slow myofiber transition. Western blotting analysis showed that RAGE is strongly re-expressed in rectus abdominis muscles derived from clinically diagnosed pancreas carcinoma patients, in both the pre-cachectic and cachectic stages, compared with healthy subjects. These data show that the overexpression of RAGE is an early event in skeletal muscles of cancer patients, and suggest an involvement of this receptor in the onset of CC. The results obtained in AgermKO mice indicate that RAGE re-expression and engagement at myofiber level drive muscle wasting and inflammation in cancer conditions. Thus, the molecular targeting of RAGE might represent a useful tool to counteract the cachectic syndrome and prolong survival in cancer patients.
38 | Effects of chronic treatment with conjugated linoleic acid on antioxidant gene expression and oxidative stress in SOD1-G93A mouse model of amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor-neuron disorder strongly linked to oxidative stress. The degenerative process gradually impairs motor-neurons function and leads to loss of homeostasis and integrity of skeletal muscle. Current therapies provide limited benefit, as the disease entails multiple pathological mechanisms. Among these, reduced activity of the nuclear factor erythroid 2–related factor 2 (Nrf2) has been observed, an effect that may compromise cellular antioxidant responses, inflammatory control, and mitochondrial biogenesis. In this preclinical study, we investigated the effects of conjugated linoleic acid (CLA), a natural and well-tolerated Nrf2 activator, by analysing the antioxidant enzymes gene expression in skeletal muscle of SOD1-G93A mice. CLA supplementation significantly enhanced the expression of key Nrf2 target genes, including G6PDX and NQO1 – which respectively encode for glucose-6-phosphate dehydrogenase and NADPH-quinone oxidoreductase – supporting NADPH production and redox balance. Additionally, CLA increased transcription of PPARGC1A, which encodes the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a known enhancer of Nrf2 transcription and pathway activation. These findings suggest that dietary CLA may strengthen antioxidant defences and offer support as a nutritional adjuvant in early ALS stages, potentially enhancing the overall effectiveness of current treatments. Funded by European Union-Next Generation EU project PRIN-PNRR 2022.
41 | SRT2104 improves muscle and cognitive outcomes and has the potential to enhance μDys gene therapy in Duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the dystrophin gene. The lack of functional dystrophin disrupts the dystrophin-glycoprotein complex, leading to progressive structural degeneration and functional impairment of myofibers. Currently, no cure is available. Although AAV-mediated micro-dystrophin (μDys) gene therapy is among the most promising strategies, its efficacy is still limited by the pathological dystrophic muscle environment, characterized by chronic inflammation and sarcolemma fragility, which compromises μDys engraftment and long-term stability. To enhance therapeutic outcomes, we are investigating a combined approach based on μDys gene therapy and pharmacological activation of Sirtuin1 (SIRT1), a NAD-dependent deacetylase involved in muscle homeostasis and regeneration. In previous work, we demonstrated that SRT2104, a clinically advanced SIRT1-activator, improves functional, metabolic, and histological parameters in mdx mice after 12 weeks of treatment. Importantly, we also observed improvements in specific neurocognitive markers, a relevant finding considering the cognitive deficits often associated with DMD. Given its broad effects on DMD-dysregulated pathways, SRT2104 represents a promising candidate to support μDys therapy by improving muscle condition and potentially allowing a reduction in viral vector dose. To explore this synergistic potential, we selected a dose of AAV9-μDys capable of restoring ≥20% of dystrophin expression, and we are currently testing different administration schedules of SRT2104 and μDys to define the optimal therapeutic window. Specifically, a time-course study is ongoing to determine the optimal timing for gene delivery following SRT2104-mediated stabilization of the muscle environment. This strategy aims not only to boost the efficacy of μDys gene therapy but also to reduce the risks associated with high AAV doses, toward a safer, more sustainable DMD treatment.
31 | Exon skipping peptide-conjugated morpholinos downregulate dynamin 2 to rescue centronuclear myopathy
Centronuclear myopathies (CNM) are rare congenital disorders characterized by muscle weakness and disorganization of myofibers. These conditions can result from dominant mutations in the DNM2 gene encoding the GTPase dynamin, making them potential targets for antisense therapy. Preclinical studies suggested decreasing DNM2 as a therapy but a recent clinical trial with antisense oligonucleotides did not effectively address the disease and showed some non-muscle toxicity. Here, to promote DNM2 downregulation in muscle versus other tissues, we used an exon skipping peptide-conjugated phosphorodiamidate morpholino (PPMO) targeting Dnm2 exon 6 splicing in the Dnm2R369W/+ mouse model for the moderate CNM form. PPMOs intravenous administration at early age (4 weeks) significantly downregulated intact (i.e. normally spliced) Dnm2 mRNA (~50%) and DNM2 protein levels in muscle. This intervention led to a rescue of muscle force, thereby preventing disease progression. PPMO administration at a later age (8 weeks), when mice demonstrated established phenotypes, efficiently decreased intact Dnm2 mRNA and protein levels in muscle, resulting in reversal of the disease phenotype and significant improvement in muscle force (from 11 mN/mg to nearly 16 mN/mg). Overall, our results indicate that PPMOs targeting Dnm2 splicing effectively decrease intact Dnm2 mRNA and protein levels in muscle and rescue muscle force in Dnm2R369W/+ mice, suggesting a promising translational approach for patients with DNM2 mutations and potentially other forms of CNM. More generally, it provides the concept of using the exon skipping strategy to decrease the protein expression of a target gene, rather than producing a shorter functional protein as is generally done.
39 | Exploring the role of the chemokine receptor CXCR2 in muscle stem cell heterogeneity
Skeletal muscle regeneration is driven by a heterogeneous population of muscle stem cells (MuSCs). This heterogeneity, particularly the non-genetic cell-to-cell variability arising from intrinsic and extrinsic factors, plays a critical role in shaping the diverse regenerative responses of this cell population. Our previous work identified surface CRIPTO protein as a key factor influencing MuSC plasticity and adaptation to the regenerative microenvironment, suggesting its involvement in generating this heterogeneity. Transcriptomic analysis of distinct CRIPTO-expressing MuSC fractions revealed a significant upregulation of the chemokine receptor CXCR2 in CRIPTO-positive cell fraction, prompting us to investigate its involvement in MuSC plasticity. CRIPTO-mediated heterogeneity involves phospholipase C (PLC) activity, which in turn regulates intracellular calcium levels. Therefore, as CXCR2 is a G-protein coupled receptor known to modulate PLC, we hypothesized the involvement of CXCR2/PLC/calcium axis in driving non-genetic heterogeneity within the MuSC population. Our results revealed variable surface expression of CXCR2 in both activated primary MuSCs and C2C12 myoblasts. Notably, isolated CXCR2positive and CXCR2negative cells displayed a dynamic interconversion, suggesting a potential role in MuSC cell plasticity. Additionally, inhibiting PLC activity in C2C12 cultures resulted in the generation of different cell populations, over time, exhibiting distinct surface CXCR2 levels as well as distinct intracellular calcium levels. Moreover, pharmacological manipulation of CXCR2 in C2C12 myoblasts resulted in altered myogenic markers profiles. These preliminary findings indicate that the dynamic expression of CXCR2 and modulation of PLC and calcium signaling may define distinct functional states within the MuSC population, suggesting their involvement in cellular metastability.
33 | Identification of mTORC1-dependent muscle-specific factors involved in neuromuscular junction maintenance
Neuromuscular junction (NMJ) dysfunction contributes to muscle wasting in numerous neuromuscular diseases such as ALS and age-related sarcopenia. Interestingly, in the last few years it has become clear that skeletal muscle fibers play a critical role to maintain a proper connection between nerve and muscle, and inhibition of muscle-specific protein synthesis leads to rapid presynaptic degeneration. mTORC1 is a key regulator of muscle protein homeostasis, and we previously showed that its activity in muscle fibers is essential to maintain NMJ structure and function. To understand the underlying mechanisms, we generated two novel tissue-specific mouse models; musclespecific RiboTag mice that allow us to observe the expression of the ribosomal protein RPL22 exclusively in skeletal muscle, and muscle-specific MetRS mice which permit the labelling and tracking of de novo synthesized muscle proteins. RiboTag mice labelling revealed increased-ribosomal protein RPL22 expression arou nd the NMJ, suggestive of increased ribosome content and, consequently, elevated translational activity. RNA sequencing of RiboTag-derived mRNA identified a strong enrichment of mTORC1 pathway components at the NMJ, suggesting a role for localized mTORC1 signaling. Furthermore, the MetRS model revealed muscle-derived proteins within the sciatic nerve and spinal cord, supporting muscle-to-nerve communication. Using musclespecific RiboTag Raptor knockout and AKT overexpression models, we observed altered translation of NMJ-related genes, reinforcing the regulatory role of mTORC1 in the NMJ translatome. Finally, we are implementing a sciatic nerve crush model to study the dynamics of muscle reinnervation and functional recovery, aiming to elucidate the contribution of mTOR signaling to this regenerative process. Together, our findings highlight the importance of mTORC1-dependent translational control in NMJ maintenance and muscle-nerve interaction.
36 | The effects of heat stress and exercise on skeletal muscle
Skeletal muscle constitutes approximately 40% of total body mass and is highly susceptible to environmental stressors. Climate change and the increasing frequency of heat waves represent a growing threat to public health. Damage due to heat stress to this tissue can therefore have widespread effects on overall health and systemic functions. However, the effects of acute heat exposure on skeletal muscle function remain poorly characterized. Indeed, several stimuli, among which heat exposure, can perturb endoplasmic/sarcoplasmic reticulum (ER/SR) homeostasis in skeletal muscle, leading to ER stress and the subsequent activation of the unfolded protein response (UPR). The aim of this study was to evaluate the impact of heat stress on in vivo muscle performance in mice and to determine whether heat exposure induces ER stress and/or activates the UPR in skeletal muscle. Additionally, with the goal of identifying strategies to protect muscle from heat-induced damage, we investigated the potential preventive role of voluntary exercise. Four experimental groups were tested: control mice (CTR), mice exposed to 39.5°C for 3 hours (heat-stressed, HS), mice allowed voluntary wheel running for 4 weeks (exercised, EX), and exercised mice subsequently exposed to heat stress (EX+HS). After 4 weeks from the start of the experiment, mice underwent functional assessment by grip strength and hanging wire tests. Gastrocnemius and soleus muscles were collected for ER stress/UPR analysis by RT-qPCR. We observed that acute heat stress significantly reduced muscle strength and increased BiP and Gadd34 mRNA levels in the soleus but not in gastrocnemius muscle. In exercised mice, these functional and transcriptional changes were prevented. Moreover, exercise alone improved muscular endurance. These findings indicate that physical exercise might represent a protective strategy to preserve skeletal muscle function under heat stress conditions.
30 | The advanced glycation end-products (ages)/rage axis drives sarcobesity, which is restrained by Vaccinium macrocarpon extract
Sarcobesity (SO) is a chronic metabolic disorder characterized by ectopic fat deposition (obesity) and concomitant progressive loss of muscle mass and strength (sarcopenia). The diffusion of a Western-like diet (WD) is considered a major risk for SO pathogenesis. WD foods rich in saturated fats and refined sugars contain high amounts of advanced glycation end-products (AGEs), a heterogeneous group of glycated proteins formed via the Maillard reaction. AGEs and their receptor, RAGE, promote ageing and several chronic diseases by sustaining inflammation and oxidative stress. We recently demonstrated that dietary AGEs induce muscle atrophy in vitro, and Vaccinium macrocarpon (VM) extract blunts AGEs. Here, we fed adult WT and RAGE-null (Ager−/−) mice with high-AGE WD for 20 weeks in the absence or presence of VM. We found AGEs accumulation in muscles and sera of WT mice, in concomitance with muscle RAGE upregulation, loss of muscle performance, reduction of myofiber areas, excessive activation of proteolytic systems, and degradation of the sarcomeric protein, myosin heavy chain (MyHC)-II. Besides muscle, WD-fed WT mice showed hepatomegaly with fat deposition and fibrosis, spleen weight increment and histological alterations, and visceral fat deposition, suggesting the establishment of a chronic low-grade inflammation causing sarcopenia. The ability of VM to abrogate dietary AGE accumulation in muscles and serum of WT mice resulted in rescue of muscle force, downregulated atrogenes and autophagy-related genes, reduced MyHC-II degradation, and restrained WD-dependent body composition changes. Remarkably, WD did not induce sarcopenia in Ager−/− mice. By in silico target fishing, potential targets of VM’s active metabolites underlying its antiglycation properties were identified. Collectively, our data unravel the AGEs/RAGE axis as a determinant mediator of SO and identify a promising natural strategy for inhibiting dietary AGE-associated sarcopenia.
24 | Smooth muscle cells from human vagina express functional transient receptor potential channels
Smooth muscle cells play a critical role in the physiology of the human vagina, especially in conditions such as the menopause, in which the decline of sex hormones levels can cause effects such as thinning of the epithelium and decreased muscle tone, impairing tissue elasticity and human vaginal smooth muscle cells (hvSMC) contractility. Elucidating the membrane proteins that regulate their activity is essential for the comprehension of the mechanisms underlying vaginal physiology and pathophysiology. Among these, Transient Receptor Potential (TRP) channels are transmembrane protein complexes acting as nonselective cation channels which can be modulated by sex hormones. They are widely expressed in several tissues, playing a pivotal role in processes as mechano-transduction, flogosis and thermal nociception. Although TRP channels have emerged as key modulators of calcium signaling and smooth muscle contractility, their effective role in hvSMCs remain largely unexplored. In this study we aimed to evaluate the expression and functionality of TRP channels in hvSMCs, considering that their activity could be disturbed following hormonal changes occurring in the menopause. We first evaluated the TRP channels expression in hvSMCs by RT-PCR and immunohistochemical analyses. The functionality of the channels was assessed by the wholecell patch clamp technique, using chemical and thermal stimuli for their activation. Our analyses showed a high expression of mRNA related to TRPV1 and TRPA1, whereas the expression of TRPM8 resulted significantly lower. The electrophysiological analyses showed that chemical and thermal stimuli were able to activate the three channels, causing an increase in current amplitude proportional to their expression. In conclusion, our results show for the first time that TRP channels are widely expressed and functional in isolated hvSMCs, and can represent a potential therapeutic target for the treatment of gynecological diseases.
21 | Transcriptional and metabolic analyses of cardiac mononucleated cells in dystrophic mdx mice upon treatment with a specific monoamino oxidase B inhibitor
Cardiomyopathy is an important cause of morbidity and mortality in Duchenne muscular dystrophy (DMD) patients. Present treatments can alleviate DMD cardiomyopathy, but there are no drugs capable of preventing cardiac tissue remodelling. Oxidative stress and inflammation are crucial players in DMD pathogenesis. The mitochondrial enzyme Monoamine Oxidase B (MAOB) is a key source of ROS, as it catalyzes the oxidative deamination of several amines yielding H2O2. In pathological conditions, its activity can overcome the cellular antioxidant defenses, altering the redox homeostasis and eliciting deleterious effects. We recently reported that targeting MAOB with specific inhibitors (iMAOB) can significantly improve skeletal muscle function and decrease inflammation and fibrosis in mdx mice. More recently, we have started to explore the therapeutic potential of iMAOB in relation to DMD cardiomyopathy, treating mdx mice at an age when cardiac disfunction is not yet clinically evident. To this purpose, three-month-old mdx mice were orally treated with iMAOB or vehicle for one month. Myeloid, endothelial and fibro-adipogenic precursor FAP cells were initially analysed via RT-PCR and we found that multiple proinflammatory and pro-fibrotic genes were downregulated by the treatment. We have now expanded our analyses by performing single-cell RNA sequencing onto mononucleated cardiac cells in treated and untreated mdx mice, as well as in wild type animals. Moreover, cardiac mononucleated cells from the same experimental conditions were also sorted by FACS into myeloid, endothelial and FAP/fibroblast sub-populations and these latter were then cultured in vitro and used to assess mitochondrial functionality via Seahorse analysis. Initial results indicate that, as expected, both approaches evidenced clear differences between wild type and mdx hearts, and suggest that the treatment with iMAOB could exert a therapeutic effect both at the transcriptional and at the metabolic level.
20 | Exercise preserves purkinje cells' integrity from paraneoplastic cerebellar degeneration in a mouse model of cachexia
Paraneoplastic syndromes are secondary conditions to the systemic effects of cancer. Among these, cancer cachexia is highly prevalent and characterized by weight loss and muscle wasting, while paraneoplastic cerebellar degeneration (PCD), defined by immune-mediated damage to Purkinje cells, is one of the most common neurological forms. Although exercise has been suggested as a strategy to counteract cachexia, the impact of training in PCD has not yet been investigated. In this study, we combined a C26 mouse model with an endurance protocol to explore the exercise effects on Purkinje cells in sedentary (SED T+) and trained (TR T+) tumour groups compared to sedentary (SED T−) and trained (TR T−) non-tumour groups. Cachexia onset was confirmed through body weight and strength loss analyses. Even if histological analysis revealed that the cerebellar area was similar across groups, Purkinje cell body circularity index and layer thickness were significantly reduced in SED T+ mice and restored in TR T+ animals. Since ZIC4 is one of the transcription factors affected by the autoimmune response in PCD, we evaluated its expression and localization both in immunohistochemistry and immunofluorescence. ZIC4 expression was reduced in SED T+ mice, but training was able to protect Purkinje cells from any autoimmune attack during tumour development. We also assessed the expression of PSD95, a common marker used for the immunolabeling of the post-synaptic side of excitatory synapses. The very low expression of the PSD95 protein in SED T+ mice suggested that the tumour mass affected the synaptic plasticity, while training again preserved synapses from the devastating effects of the tumour circulating factors. Altogether, these results suggest an important role of training in the protection of cerebellum damage. Further studies are needed to clarify the role of exercise in protecting the stimulating synapses of the efferent fibers of the cerebellar cortex.