CU TRANSPORTER ATP7A AND EXTRACELLULAR SOD FUNCTION IN ADAPTIVE ANGIOGENESIS AND REGENERATION OF SKELETAL MUSCLES IN RESPONSE TO EXERCISE OR INJURY: ROLE OF EXOSOMES

Abstract

The overall goal of this dissertation is to elucidate novel mechanisms by which communication of skeletal muscle (SKM) and endothelial cells (ECs) through “exosomes” regulates adaptive angiogenesis and regeneration of SKM in response to physical exercise in type 2 diabetes mellitus (T2DM) (project 1) or injury (project 2) by focusing on the Copper (Cu) transporter ATP7A-extracellular SOD (ecSOD, SOD3) pathway. In project 1, we investigated the role of exercise-induced angiogenic effects on ECs in T2DM. We isolated plasma exosomes from control, T2DM and SOD3-/- mice with two weeks of voluntary wheel exercise using differential ultracentrifugation. Isolated exosomes were characterized by ZetaView, TEM and exosome markers (CD63 and Tsg101). Both SOD3 and ATP7A proteins were significantly induced in plasma exosomes by exercise in both mice and humans. Plasma exosomes from T2DM and SOD3-/-/T2DM sedentary mice impaired ECs angiogenic function compared to control mice, which were restored by exercise in only T2DM but not SOD3-/-/T2DM mice. Furthermore, exosomes overexpressing SOD3 significantly enhanced angiogenesis in ECs by increasing local H2O2 levels in a heparin binding domain-dependent manner and restored wound healing and angiogenesis in T2DM or SOD3-/- mice. In project 2, we investigated the role of Cu transporter ATP7A in myogenic differentiation and skeletal muscle regeneration upon injury. C2C12 myoblasts differentiation to myotubes was associated with an increase in ATP7A and SOD3 expression in cell lysates and exosomes isolated from conditioned media. ATP7A depletion with shRNA in myoblasts significantly inhibited myogenic differentiation and angiogenic effects of conditioned media-derived exosomes. Mechanistically, silencing SOD3, chelating Cu by TTM or inhibiting lysyl oxidase (LOX) activity by BAPN significantly inhibited myogenic differentiation, suggesting that ATP7A-SOD3/LOX axis is required for Cu-dependent myogenesis. Cardiotoxin-induced SKM injury model reveals that ATP7A expression was increased in activated, but not quiescent Pax7-positive satellite cells in injured SKM. Functionally, both Cu transporter defective ATP7A mutant and SOD3-/- mice showed impaired SKM regeneration, diminished myofiber sizes, decreased number of centralized myonuclei and satellite cells. In conclusion, our study reveals a novel role of Cu transporter ATP7A and exosomal SOD3 in promoting adaptive angiogenesis and SKM regeneration serving as a novel promising therapeutic tool for resolving impaired angiogenesis.