Dear Editor, Collagen VI (ColVI)-related myopathies are a distinct group of progressive muscle disorders, which include Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM), for which no therapy is yet available. In the last two decades, much effort was spent on the elucidation of the pathogenic mechanisms underlying ColVI-related myopathies, also by taking advantage of the ColVI null (Col6a1−/−) mouse model,1 in order to identify druggable targets for prospective therapies.2-4 The results obtained within this work provide a proof-of-concept for the repurposing of the Food and Drug Administration (FDA)-approved salbutamol in the context of ColVI-related myopathies, as they demonstrate that systemic salbutamol administration can recover the major structural and functional neuromuscular junction (NMJ) defects occurring in murine muscles downstream ColVI deficiency, and previously described also in patients.5 We selected salbutamol for three main reasons: (i) it was previously reported to ameliorate NMJ structure and electrophysiological cues in animal models of human NMJ-related disorders; (ii) it is an FDA-approved drug already used with success as an off-label medication in several diseases displaying not only primary but also secondary NMJ defects, including Pompe disease, spinal muscular atrophy and Duchenne, Becker and facioscapulohumeral muscular dystrophies; (iii) it was proven to be safe in the majority of the open clinical trials and therefore eligible for future rapid translational applications in the clinical settings.6 Five-month-old wild type (WT) and Col6a1−/− male mice were treated with either vehicle or salbutamol at two different doses, 4 and 8 mg/kg/die, subcutaneously administered via Alzet osmotic minipumps for 28 days (Figure S1 and Supporting Information). First, we investigated the impact of salbutamol on NMJ structural defects and found that the abnormally increased NMJ fragmentation displayed by vehicle-treated Col6a1−/− diaphragms, compared to WT animals (Figure 1A–C), was recovered by treatment with salbutamol at the higher dose (Figure 1D–F). Furthermore, morphometric analysis of NMJs showed that both low- and high-dose salbutamol induced a significant increase in postsynaptic terminal areas and compactness in Col6a1−/− mice (Figures 1G–L and S2A), coherently with reports of salbutamol beneficial effects in expanding NMJ size in animal models of congenital myasthenic syndromes (CMS).7, 8 The analyses performed on treated WT mice confirmed the general trend of salbutamol in increasing NMJ size (Figure S2B–H). To evaluate whether salbutamol also ameliorated neuromuscular transmission, we first monitored the four-limb hanging test performance. Besides confirming a marked difference between WT and Col6a1−/− mice (Figure 1M), the test highlighted an improved performance of Col6a1−/− mice treated with high-dose salbutamol (Figure 1N,O). Moreover, ex vivo electrophysiological recordings performed in diaphragm muscles revealed that high-dose salbutamol treatment recovered the NMJ functional parameters found to be altered in Col6a1−/− mice, when compared to WT animals, including a lower amplitude of nerve-evoked endplate potentials and of miniature endplate potentials (mEPP), a reduced quantal content and input resistance, as well as decreased endplate currents, miniature endplate currents and mEPP frequency (Figure 1P–V). Notably, assessment of neuromuscular transmission by repetitive stimulation of the phrenic nerve for 25 pulses at 5 Hz further highlighted that the higher decrement registered upon vehicle treatment in Col6a1−/− mice, when compared to WT animals, was also rescued by salbutamol (Figure 1W), clearly indicating that high-dose salbutamol treatment not only ameliorates NMJ fragmentation but also elicits NMJ functional improvement in Col6a1−/− mice. Since β2-agonists are well-known anabolic agents,9 we hypothesised that Col6a1−/− muscles could benefit per se from salbutamol treatment. Thus, we evaluated salbutamol impact on muscle mass, attesting a significant increase of body weight and of the absolute and normalised tibialis anterior (TA) and gastrocnemius muscle weight both in Col6a1−/− and WT mice (Figures 2A–C and S3A–F). Consistently, morphometric analysis of TA cross-sections revealed a significant increase in average cross-sectional area and minimum Feret's diameter elicited by high-dose salbutamol treatment in Col6a1−/− mice (Figure 2E,F), paralleled by a shift of myofibre size distribution towards larger myofibre classes (Figure 2G–L) and by a reduction in myofibre density (Figure 2M). A similar shift in fibre size was detectable in salbutamol-treated WT muscles (Figure S3G–O), whereas myofibre density was not significantly affected (Figure S3P). Interestingly, while salbutamol treatment in either Col6a1−/− and WT mice did not influence the occurrence of centronucleated fibres (Figures 2N and S3Q), a higher proportion of regenerating fibres, together with increased Myh3 transcript levels, was detectable in muscles of both genotypes upon high-dose treatment (Figures 2O–Q and S3R–T). Such results validated the concept that salbutamol not only promotes muscle remodelling in WT animals but also counteracts muscle mass loss and promotes muscle regeneration in Col6a1−/− mice. In keeping with literature, showing that β2-agonists can induce slow-oxidative to fast-glycolytic myofibre switch in rodents,9 despite the absence of any overt change in succinate dehydrogenase staining in WT and Col6a1−/− muscles (Figures 3A,B and S4A,B), an upregulation of Myh4 gene (Figures 3C,D and S4C,D) and an increased percentage of type IIB fibres (Figure 3E,F) were detected upon salbutamol treatment in Col6a1−/− mice, compared to the corresponding vehicle-treated mice. Similar changes were not detectable in salbutamol-treated WT muscles (Figure S4E,F). We then evaluated salbutamol's ability to increase muscle strength, by performing in vivo tetanic force measurements on gastrocnemius muscles of WT and Col6a1−/− mice, following either salbutamol or vehicle administration. The impaired muscle strength of Col6a1−/−, compared to WT mice, both in terms of absolute and normalised force (Figure 4A,B), resulted ameliorated by salbutamol since it induced a significant gain in the absolute force, mostly evident at intermediate stimulation frequencies (Figure 4C), which, notably, remained significant even when normalised to muscle weight upon high-dose treatment (Figure 4D). In WT mice, treatments determined a significant gain only in absolute force at intermediate frequencies of stimulation (Figure S5A,B). Of note, the curves corresponding to the normalised force of vehicle-treated WT mice displayed a complete overlap with those of salbutamol-treated Col6a1−/− mice at intermediate stimulation frequencies (Figure S5C,D), demonstrating that salbutamol induces a substantial amelioration of in vivo muscle strength. Finally, β2-adrenergic receptor protein levels were significantly reduced in both TA and diaphragm muscles of Col6a1−/− mice after the 4-week salbutamol treatment at both doses (Figure 4E,F,H,I), consistently with receptor desensitisation9 and with a potential undesired mitigation of the biological response to the treatment. Considering that salbutamol is a sympathomimetic drug and supposing it might reconstitute a defective sympathetic innervation of NMJs of Col6a1−/− mice, we monitored tyrosine hydroxylase (TH) as a marker for sympathetic neurons. TH protein levels appeared only mildly affected in TA muscles (Figure 4E,G), while in diaphragms, a decrease in TH levels was detectable in high-dose salbutamol-treated Col6a1−/− mice, when compared to vehicle-treated ones (Figure 4H,J). In situ quantification of NMJs presenting sympathomimetic innervation did not highlight any further difference between vehicle-treated WT and vehicle- or high-dose salbutamol-treated Col6a1−/− mice (Figure 4K,L), and norepinephrine concentration in serum was not affected as well by the different treatments (Figure S6). Therefore, we cannot infer that salbutamol efficacy relies on an overtly impaired sympathetic innervation in Col6a1−/− mice. In conclusion, our results show that 1-month systemic treatment with salbutamol stabilises NMJ structures while ameliorating the myopathic phenotype of Col6a1−/− mice. By limiting muscle wasting and improving neuromuscular transmission in mice, salbutamol administration may provide clinical benefits for patients. Of note, salbutamol doses adopted here are comparable to those proven to be successful in counteracting CMS phenotype in murine models.8 On the other hand, a direct conversion to human application, where the highest effective dosages used in clinics for CMS adult patients range between 6 and 12 mg per die,10 is not realistic. In addition, subcutaneous infusion for salbutamol administration is less frequently opted in patients, where the per os route is preferred in the context of CMS and other dystrophies.6 Nonetheless, being salbutamol already approved by the FDA, optimised salbutamol-based therapeutic strategies might be more rapidly translated into clinical trials, an aspect of high relevance in the clinical setting for BM/UCMD patients. Matilde Cescon designed the study, acquired funding and supervised analysis; Sonia Calabrò and Matilde Cescon performed in vivo treatments; Sonia Calabrò performed in vivo and ex-vivo analysis; Leonardo Nogara performed in vivo muscle force measurements; Yongzhi Jian and Manuel Valentin performed ex-vivo electrophysiological analysis; Dario Bizzotto and Paola Braghetta managed mouse colonies; Loris Russo and Lisa Gambarotto contributed to the analysis; Bert Blaauw and Said Hashemolhosseini supervised in vivo and ex-vivo electrophysiological analysis, respectively; Paolo Bonaldo provided Col6a1−/− mice and acquired funding; Sonia Calabrò and Matilde Cescon wrote the original draft; all the other authors contributed to revision of the manuscript. We acknowledge the light microscopy facility of the Biology Department of the University of Padova. This work was funded by AFM Telethon (Trampoline Grant Number #22360 to Matilde Cescon), University of Padova (PRID-DMM to Matilde Cescon); Telethon Foundation (Grant GGP19229 to Paolo Bonaldo), and the Italian Ministry of Education, University and Research (Grant P2022Y2A3L funded in the framework of NRRP, Mission 4.2, Investment 1.1 "progetti di ricerca di Rilevante Interesse Nazionale - PRIN", funded by the European Union - Next Generation EU, CUP C53D23007520001, to Matilde Cescon, Grant P20227YB93 to Matilde Cescon; Grant 201742SBXA to Paolo Bonaldo), German Research Council (DFG) Grants (HA3309/3-1, HA3309/6-1 and HA3309/7-1 to Said Hashemolhosseini). Moreover, as part of the activities of the National Center for Gene Therapy and Drugs based on RNA Technology, funded in the framework of the National Recovery and Resilience Plan (NRRP), Mission 4 "Education and Research", Component 2 "From Research to Business", Investment 1.4 "Strengthening research structures for supporting the creation of National Centres, national R&D leaders on some Key Enabling Technologies", this work was funded by the Europena Union - Next Generation EU, Project CN00000041, CUP B93D21010860004, Spoke n. 5 "Inflammatory and infectious diseases". The authors declare no conflicts of interest. Animal procedures were approved by the Animal Ethics Committee of the University of Padova and authorised by the Italian Ministry of Health (Project No. 98/2020-PR). All data needed to evaluate the conclusions in the paper are present in the paper and the Supporting Information. 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