"To accomplished the first objective, an extension of the previous pilot study was started before the beginning of the project. The performed work included included colony management, in vivo test, mice dissection and analysis of samples. The main part of this first aim was published on the paper ""Tamoxifen prolongs survival and alleviates symptoms in mice with fatal X-linked myotubular myopathy"" on Nature Communications journal, with Gold Open Access. Summarizing the main results, as it was observed on the pilot study, lifespan of treated KO mice was expanded compared to the not treated ones, clinical stage ameliorated and grid test score (in vivo test to measure muscle performance) of KO treated mice was close to WT values. Tamoxifen treatment also improved the force generated by the triceps surae, a large muscle group of the lower leg that makes up most of the calf volume and contains gastrocnemius, plantaris and soleus muscles. Tamoxifen enhanced the absolute and specific phasic and tetanic force of KO treated mice. Histology analyses were performed and it showed how tamoxifen treatment reduced by 50% the number of myofibers showing nuclei in abnormal position (a major histological feature of XLMTM). Muscle samples from WT, KO and KO treated mice allowed us to analyze the consequences of tamoxifen treatment on muscular transcript and protein levels of selected targets for their known contribution to XLMTM pathogenesis. In general, tamoxifen helps to normalize molecular hallmarks of the disease.
The second aim of the project was the pre-clinical study of tamoxifen treatment with other compounds. After the good results of tamoxifen extending lifespan, combination therapy seemed to be the next step to prevent leg muscle atrophy and achieve complete functional rescue. The selected compounds were L-citrullin and IGF. L-citrullin is used by body-builders to increase muscle mass. To determine the optimal dose for mice, a pilot study was carried out and 3 mg/kg/day was choses as the minimum dose having beneficial effects on muscle mass. This compound can be added to the food and taken orally without manipulation of the mice. A modified version of IGF-1, growth factor similar to insulin, was the second compound. It stimulates proliferation and differentiation of satellite cells during muscle regeneration. Increasing IGF-1 levels on animal models affected by loose and degradation of muscle fibers has shown beneficial effects. For the moment, only a pilot study with few animals per group has been carried out. Summarizing, animals were followed-up during the whole treatment (from 21 to 45/90 day-old depending on the group). Mice weight and clinical state were determined twice a week and grid test, once a week. When arriving at the stablished time point, final measurements of respiratory function (plethysmography), body composition (echo-MRI), bone density and kyphosis (micro-CT) were performed. In general, no differences were observed between obtained results from mice treated with tamoxifen and those treated with a combined treatment. Tamoxifen combined with L-citrullin seemed to increase muscle mass in comparison with the other treatments at 45-days-old. This effect was only noticeable after muscle dissection and sample weighting. The results for bone density and the kyphose are still being analyzed."
