Sara Menazza, Bert Blaauw, Tania Tiepolo, Luana Toniolo, Paola Braghetta, Barbara Spolaore, Carlo Reggiani, Fabio Di Lisa, Marc

Several studies documented the key role of oxidative stress and abnormal production of reactive oxygen species (ROS) in the pathophysiology of muscular dystrophies (MDs). The sources of ROS, however, are still controversial as well as their major molecular targets. This study investigated whether ROS produced in mitochondria by monoamine oxidase (MAO) contributes to MD pathogenesis. Pargyline, a MAO inhibitor, reduced ROS accumulation along with a beneficial effect on the dystrophic phenotype of Col6a1 mice, a model of Bethlem myopathy and Ullrich congenital MD, and mdx mice, a model of Duchenne MD. Based on our previous observations on oxidative damage of myofibrillar proteins in heart failure, we hypothesized that MAO-dependent ROS might impair contractile function in dystrophic muscles. Indeed, oxidation of myofibrillar proteins, as probed by formation of disulphide cross-bridges in tropomyosin, was detected in both Col6a1 and mdx muscles. Notably, pargyline significantly reduced myofiber apoptosis and ameliorated muscle strength in Col6a1 mice. This study demonstrates a novel and determinant role of MAO in MDs, adding evidence of the pivotal role of mitochondria and suggesting a therapeutic potential for MAO inhibition. 2 by gest on Sptem er 7, 2016 ht://hm g.oxfournals.org/ D ow nladed from Introduction Muscle dystrophies (MDs) are a heterogeneous group of inherited human disorders primarily affecting skeletal muscles. Typically, MDs show diffuse wasting and weakness of muscles, associated with degeneration and regeneration of muscle fibers. Duchenne muscular dystrophy is the most common and severe form of MD worldwide. This progressive and lethal X-linked myopathy is characterized by deficiency of dystrophin, a subsarcolemmal protein critical in membrane stabilization and prevention of contraction-induced cell membrane damage (1). Although mutations in components of the dystrophin glycoprotein complex are responsible for the most devastating MDs, mutations of genes coding for extracellular matrix proteins play a causative role in other forms of MDs. Indeed, another group of MDs is due to inherited mutations in genes encoding collagen VI. Collagen VI is a major component of the endomysium, where it is localized just outside the basement membrane of muscle fibers. Mutations of Collagen VI cause two muscle diseases in humans, namely Bethlem myopathy (MIM#158810) and Ullrich congenital muscular dystrophy (MIM#254090) (2). Bethlem myopathy is a relatively mild and slowly progressive myopathic disorder, whereas UCMD is a severe and rapidly progressive muscle disease usually causing early death due to respiratory failure. Several studies documented the key role of oxidative stress and abnormal production of ROS in the pathophysiology of MDs (3-6). Antioxidant treatments have been shown to counteract myocyte injury in mdx mice demonstrating the key role of reactive oxygen species (ROS) in MD pathogenesis (7). Indeed, ROS formation appears a causative event rather than a consequence of muscle degeneration (8). Mitochondria are generally indicated as a major source of ROS. In fact, recent studies provided clear evidence of the crucial role of mitochondria in MDs. This concept is supported by genetic or pharmacological reduction of the open probability of the mitochondrial permeability transition pore, 3 by gest on Sptem er 7, 2016 ht://hm g.oxfournals.org/ D ow nladed from that has been demonstrated to prevent myofiber injury characterizing experimental models of MDs (911). Besides the respiratory chain, a relevant source of ROS in mitochondria is represented by monoamine oxidases (MAO). These flavoproteins, which exist in two isoforms, MAO-A and MAO-B, are located in the outer mitochondrial membrane and catalyze the oxidative deamination of neurotransmitters and dietary amines, generating hydrogen peroxide (12). The monoamine catabolism generates aldehydes, ammonia and hydrogen peroxide. MAO has been extensively studied at the level of the central nervous system while their role in other tissues has been scarcely investigated. Nevertheless, evidence has been provided that MAO contributes to cardiac diseases (13,14). As far as the targets of ROS are concerned, we hypothesized the myofibrillar proteins could represent important intracellular targets of ROS involved in contractile dysfunction of the dystrophic muscle. Indeed, in several models of myocardial dysfunction, such as coronary microembolization and heart failure, we demonstrated that myofibrillar proteins, such as tropomyosin (Tm), actin and desmin were modified by ROS and that the contractile impairment was related to formation of disulphide cross-bridges in Tm (15,16). In the present study we investigated the role of MAO in skeletal muscles of two murine models for different forms of MDs, namely Col6a1 and mdx mice. Our findings demonstrate that accumulation of ROS related to MAO activity plays a pivotal role in both loss of cell viability and contractile derangements of dystrophic skeletal muscles. This is supported by the protective efficacy of the MAO inhibitor pargyline on oxidative stress, alterations of muscle structure and function, and loss of cell viability in the two murine MD models. 4 by gest on Sptem er 7, 2016 ht://hm g.oxfournals.org/ D ow nladed from