Role of oxidative stress in the modulation of muscle homeostasis and therapeutic approach by antioxidants delivered by targeted liposomes

Research project

Description

Objective. The objectives of this proposal are to elucidate the molecular mechanisms triggered by excessive oxidative stress in skeletal muscle and to develop an innovative liposome-based delivery system of antioxidants into mitochondria, to reduce oxidative stress and restore muscle homeostasis.
Rationale. Homeostasis represents an important and critical parameter of adult skeletal muscle and is defined as the capability to maintain a normal structure and function in a dynamic equilibrium. How oxidative stress contributes to guarantee or to alter this internal balance is still not fully elucidated. Sustained oxidative stress has been associated with many pathological conditions and it is now widely considered a major contributor for the imbalance between muscle protein degradation and synthesis. On the one hand, increased oxidative stress triggers multiple catabolic signaling in skeletal muscle, such as autophagy and ubiquitin-proteasome pathways; on the other hand, it hampers the myogenic potential of satellite cells, the progenitor cells responsible for adult muscle growth and renewal. Our attention will be focused on studying molecular mechanisms underlying the response to oxidative stress in neurogenic muscle atrophy - either following sciatic nerve rescission or in Amyotrophic Lateral Sclerosis (ALS) - and aging. Even if neurogenic muscle atrophy and aging seem to be unrelated conditions and distant from each other, they share high level of oxidative stress, progressive muscle weakness and atrophy. Reducing the levels of ROS, from high to normal, in skeletal muscle may be a useful therapeutic approach to ameliorate many different pathological conditions characterized by excessive oxidative stress. Reduction of ROS level can be achieved by different strategies, among which the administration of antioxidants. The possibility of a specific delivery of antioxidants directly into mitochondria, the main source of ROS in the cell, should significantly improve their therapeutic efficiency with respect to a non-specific administration and should reduce the onset of undesired side effects. Different strategies have been explored for a specific delivery to mitochondria, all being hampered by the very special morphology of this organelle. Among them, liposomes have been shown to be very promising because of their biocompatibility, versatility, and high loading efficiency of both hydrophobic and hydrophilic molecules or macromolecules.
Research plan. Histone Deacetylase 4 (HDAC4) activity and expression is induced in skeletal muscles in response to denervation and controls skeletal muscle mass and innervations. We will investigate the connections between the molecular pathways triggered by ROS and HDAC4 following denervation or in a mouse model of ALS, by using mice lacking HDAC4 in skeletal muscle (HDAC4mKO). We plan to identify the molecular mediators regulated by HDAC4 in the ROS-mediated atrophy response, by analyzing the mitochondria network, the amount of oxidative stress and the catabolic pathways activated by ROS in HDAC4mKO mice. We expect that the absence of HDAC4 will reduce oxidative stress following denervation and we will protect skeletal muscle from the progressive degeneration and weakness induced by ALS.
Because impairment in muscle regeneration sustains the atrophic state, promoting muscle repair and growth has been shown to be beneficial in many pathological conditions of muscle atrophy, including ALS. Therefore, we intend to investigate the myogenic potential of satellite cells from HDAC4mKO mice following denervation and in ALS, also in relation to the signaling of receptor for advanced glycation endproducts (RAGE), which is a pro-myogenic factor and an indirect target of HDAC4. We expect RAGE to be upregulated in HDAC4mKO satellite cells and this may account for a better myogenic potential in the presence of increased oxidative stress. RAGE transduces a pro-myogenic signal via activation of a Rac1-cdc42/MKK6/p38 MAPK/myogenin pathways and muscles lacking RAGE show remarkably delayed regeneration upon injury. Moreover aged human satellite cells show truncated, signaling deficient, RAGE protein and higher levels of S100B, which exerts anti-myogenic effects via activation of IKKbeta/NF-kB. Whether RAGE and/or intracellular S100B are target of oxidative stress and/or are active players in the generation of oxidative stress in myoblasts is not known. Moreover, oxidative stress activates the transcription factor Nrf2 that directly regulate the expression of antioxidant/detoxifying genes and, indirectly, inhibits NF-kB. In this project, we intend to define the role of RAGE and S100B in the oxidative stress conditions focusing on the regulation of RAGE and S100B expression, Nrf2 and NF-κB activity, myostatin and TNF-alpha pathways. Moreover, RAGE and S100B pathways and functions will be investigated in satellite cells under pathological conditions characterized by elevated ROS levels: following denervation, in a mouse model of ALS, and in elderly human subjects. We will analyze the effects of S100B and RAGE inhibition on myogenic potential and the effects of antioxidants, inhibitors of NF-kB and/or pharmacological activators of Nrf2, in the restoration of physiological RAGE and S100B levels and in the effects on myogenic differentiation. Understanding the mediators of ROS-mediated muscle atrophy will provide putative targets for the development of new therapies. An additional focus of this proposal is to define the correlation between ROS levels and calcium homeostasis, mitochondrial functionality and myogenic process in human satellite cells. Measurement of ROS levels, mitochondrial membrane potential, cytosolic and mitochondrial calcium - in the presence or not of oxidant, or antioxidant - will be correlated with satellite cell ability to differentiate. We expect that impairment in myogenic potential is associated with increased intracellular calcium and elevated ROS mitochondrial levels in elderly satellite cells. Therefore, delivering antioxidant directly into muscle mitochondria may be an high-potential therapeutic approach to significantly improve different muscle pathologies characterized by high levels of oxidative stress.
To this aim, we plan to develop an innovative liposome-based drug delivery systems (DDS) able to specifically target both the skeletal muscle cells and the mitochondria therein and to deliver antioxidants (AO), resveratrol (RSV) and ubiquinone-10 (COQ10) with high efficiency in these intracellular organelles. The DDS will be designed to be highly specific for the skeletal muscle cell and for mitochondria, to increase the local bioavailability of AO, and to avoid side effects. This DDS is, hence, in principle eligible for a systemic route of administration. To address this objective, we will realize a composite core-shell DDS, where the shell will be composed of biocompatible polymers (BC), as dextran and chitosan, functionalized with proper molecules, able to target the skeletal muscle cell and the core will be a liposome formulated with mitochondriotropic cationic Bolalipids. The AO will be encapsulated in the liposome core. Besides the targeting to the cell, the BC is supposed to help the liposomes to reach undamaged the cytosol and, then, deliver AO to mitochondria. RSV and COQ10 have shown high potential in the treatment of disorders mediated by oxidative stress, however, they displays low solubility and stability in water, and RSV also some cytotoxicity. Their entrapment in targeted liposomes might improve their bioavailability and therapeutic efficiency.
StatusActive
Effective start/end date1/1/12 → …

Funding

  • MIUR - FIRB

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Liposomes
Oxidative Stress
Homeostasis
Antioxidants
Muscles
Skeletal Muscle
Histone Deacetylases
Amyotrophic Lateral Sclerosis
Muscular Atrophy
Denervation
Mitochondria
Drug Delivery Systems
Muscle Mitochondrion
NF-kappa B
Calcium
Skeletal Muscle Satellite Cells
Organelles
Biological Availability
Regeneration
Polymers