Methods for Replicating P188-Induced Sarcolemma Stabilization in Dystrophin-Deficient Skeletal Muscle Based on Length Kinetics

Abstract

Duchenne Muscular Dystrophy (DMD) is a severe muscle-wasting disease brought about by a genetic mutation that prevents the synthesis of dystrophin, a protein necessary for myofiber cell membrane integrity. There is no known method capable of curing DMD, although many therapies have been developed to curb its symptoms and delay its progression. One such solution is the application of synthetic copolymers as a substitute for dystrophin. A recent study investigated the therapeutic possibilities of poloxamer 188 (P188), a linear, amphiphilic triblock copolymer, by observing twitch kinetics of sarcomere length in vitro (Hahn et al., 2023). They found that P188 can effectively improve contractility in dystrophic skeletal muscle. Our experiment initially aimed to follow up on this study by using a similar approach for testing bottlebrush copolymers. However, when practicing the procedure with P188, we found it difficult to replicate Hahn et al.’s results. Hence, we adjusted the methodology in various ways to determine the most accurate and precise technique before moving on to new experimental conditions. We measured length kinetics from different regions of a myofiber and used centrifugation to shorten the procedure length. Because Hahn et al. used Fura-2AM as a calcium indicator, we also tried implementing it in attempt to more accurately follow their procedure. We found that using centrifugation and measuring contractility from the myofiber’s center produces results closest to their study. Despite this, our study showed a less marked improvement in myofiber peak contraction height. Further refinement of our methods is required to improve our technique’s reliability and efficiency before we can proceed to trials with bottlebrush copolymers.