Abstract P1063: Investigation Of Synthetic Membrane-stabilizing Copolymers To Dystrophin-deficient Skeletal Muscle Fibers

Abstract

Duchenne muscular dystrophy (DMD) occurs by the lack of cytoskeletal protein dystrophin which is essential for the preservation of structural integrity of the muscle cell membrane. DMD develops severe skeletal muscle weakness, degeneration, and early death. We applied amphiphilic synthetic membrane stabilizers to the mdx skeletal muscle (flexor digitorum brevis; FDB) to compare the effectiveness in restoring contractile function in dystrophin-deficient muscle. After isolating the FDB fibers by enzymatic digestion and trituration from twenty-six adult male mice (5 C57/BL10, 21 mdx; at least 15 fibers/animal), we plated them on laminin-coated coverslip and treated them with poloxamer 188 (PEO 75 -PPO 30 -PEO 75 ; 8,600 g/mol; P188), architecturally reversed (PPO 15 -PEO 200 -PPO 15 , 10,700 g/mol), and diblock (PEO 75 -PPO 15 -C 4 , 4,200 g/mol) copolymers in 15 and 150 μM at 25 o C for 10 min. We assessed the twitch kinetics of sarcomere length (SL) and intracellular Ca 2+ transient by Fura-2, AM in field stimulation (25 V, 0.2 Hz, 25 o C). Twitch peak SL shortening of mdx FDB fibers was depressed to 30% of the dystrophin-replete control FDB fibers from BL10 (P < 0.001). Treatment of mdx single FDB fibers with copolymers rapidly restored the twitch peak SL shortening by over 60%, especially P188 at 15 μM (60% of BL10 FDB, P < 0.001) and 150 μM (90% of BL10 FDB, P < 0.001). Twitch peak Ca 2+ transient from mdx FDB fibers was depressed by 50% compared to BL10 FDB fibers (P < 0.001). Interestingly, acute copolymer treatment of mdx FDB fibers significantly increased the twitch peak Ca 2+ transient by 20-45%, especially P188 at 150 μM up to 75% of the BL10 FDB fibers (P = 0.472). Our data shows that the structure and concentration of the synthetic copolymers lead to different effectiveness on rescuing the twitch contractile function and Ca 2+ handling in dystrophin-deficient skeletal muscle fibers. Notably, acute treatment for 10 min with copolymers (P188, 150 μM) elicited over 3-fold improvement of peak SL shortening in mdx isolated skeletal muscle fibers. This study highlights copolymers as effective membrane stabilizing molecules in the restoration of dystrophin-deficient skeletal muscle function and the importance of copolymer structure, molecular weight, and concentration.