Epigenetic modulation and fibrosis: towards new targets for innovative therapies in Duchenne muscular dystrophy

Duchenne Muscular Dystrophy (DMD) is a rare, degenerative disease caused by the absence of protein dystrophin in myofibers. This leads to a complex pathogenetic cascade, not yet fully clarified, which ends in loss of muscle tissue. No definitive cure is available, and pre-clinical are pivotal to identify innovative therapeutic strategies.In this PhD thesis I focused my attention on the interplay between inflammation, mechanical-metabolic uncoupling and aberrant epigenetic control in promoting skeletal muscle fibrosis in DMD, with the aim to identify novel druggable targets and approaches: a) To clarify the role of Liver kinase B1 (LKB1) in dysmetabolic conditions and its interplay with histone deacetylases (HDAC) deregulation. Firstly, we evaluated, by biochemical approaches, the expression of LKB1 and related proteins, in gastrocnemious (GC) muscles of BL-10 mdx and D2-mdx mice at different stages of pathology and/or under the action of stressors (exercise). Then, to assess the potential epigenetic control of LKB1 activity, we performed biochemical analyses in GC muscles of D2-mdx chronically treated with histone deacetylases inhibitors (HDACi). b) To evaluate the effect of L-citrulline administration. The underlying hypothesis is that L-citrulline, as nitric oxide (NO) donor, could restore NO levels, altered in dystrophic myofibers, and consequently the NO-dependent inhibition of HDAC. L-citrulline was administered to 4-5-week-old mdx mice at a final dose of 2 mg/g per day (16.5 g L-citrulline/kg diet) for 8 weeks. The outcomes were evaluated by using a multifunctional approach, including in vivo indices of muscle function and ex vivo functional, biochemical and histological readouts. c) To clarify the role of complement system in DMD pathogenesis, with a focus on regeneration and fibrosis. We performed Real time- PCR and histological analysis in BL10-mdx and D2-mdx at different stages of pathology and after treatment with two compounds able to reduce inflammation and fibrosis, such as a-methylprednisolone (PDN) or JMV2894 (a synthetic mimetic of ghrelin) d) To assess the potential benefit of a novel antibody targeting latent TGFbinding protein (LTBP)-4 on disease signs of dystrophic mice, considering that TGFb appears to be a key player of final fibrosis in DMD. Then, during my stage at the pharmaceutical industry Italfarmaco, we test the murine antibody anti-LTBP4 (mAb 88), for 24 weeks (20mg/kg 5 times a week, intraperitoneally) to 7-week-old BL-10 mdx and age-matched D2-mdx mice. The efficacy of treatment was evaluated via a multidisciplinary approach based on in vivo and ex vivo readouts. Results. a) we observed a marked reduction of LKB1 and related proteins expression in both dystrophic mice. Importantly, a restoration in was observed in D2-mdx mice treated with HDACi; b) L-Citrulline administration guaranteed a recovery of muscle strength and function, particularly in diaphragm muscle and a decrease in fibrosis was also observed; c) complement system appears to be overactivated in both strains of dystrophic mice respect to control ones, mainly in the acute stage of pathology. However, a further increase in C1q component complement expression was found in mice treated with anti-inflammatory and anti-fibrotic drugs, suggestion a double-edged sword role of this system; d) surprisingly, the anti-ltbp4 treatment exerted minor effects on fibrosis suggesting that this approach still needs to be optimized This present work provided to evaluate new druggable targets in dysmetabolism, disclosing in particular a key and reciprocal role of LKB1-mediated pathways in epigenetic modulation, while L-citrulline may represent an important adjuvant as dietary supplement for its benefit on key pathology signs. However, the development of effective and specific antibodies against LTBP4 needs further confirmation, likely for the induction of compensatory neutralizing immune-mediated