Mechanical factors tune the sensitivity of mdx muscle to eccentric strength loss and its protection by antioxidant and calcium modulators

Abstract

Background: Dystrophin deficiency sensitizes skeletal muscle of mice to eccentric contraction (ECC)-induced strength loss. ECC protocols distinguish dystrophin-deficient from healthy, wild type muscle, and test the efficacy of therapeutics for Duchenne muscular dystrophy (DMD). However, given the large lab-to-lab variability in ECCinduced strength loss of dystrophin-deficient mouse skeletal muscle (10-95%), mechanical factors of the contraction likely impact the degree of loss. Therefore, the purpose of this study was to evaluate the extent to which mechanical variables impact sensitivity of dystrophin-deficient mouse skeletal muscle to ECC. Methods: We completed ex vivo and in vivo muscle preparations of the dystrophin-deficient mdx mouse and designed ECC protocols within physiological ranges of contractile parameters (length change, velocity, contraction duration, and stimulation frequencies). To determine whether these contractile parameters affected known factors associated with ECC-induced strength loss, we measured sarcolemmal damage after ECC as well as strength loss in the presence of the antioxidant N-acetylcysteine (NAC) and small molecule calcium modulators that increase SERCA activity (DS-11966966 and CDN1163) or lower calcium leak from the ryanodine receptor (Chloroxine and Myricetin). Results: The magnitude of length change, work, and stimulation duration ex vivo and in vivo of an ECC were the most important determinants of strength loss in mdx muscle. Passive lengthening and submaximal stimulations did not induce strength loss. We further showed that sarcolemmal permeability was associated with muscle length change, but it only accounted for a minimal fraction (21%) of the total strength loss (70%). The magnitude of length change also significantly influenced the degree to which NAC and small molecule calcium modulators protected against ECC-induced strength loss. Conclusions: These results indicate that ECC-induced strength loss of mdx skeletal muscle is dependent on the mechanical properties of the contraction and that mdx muscle is insensitive to ECC at submaximal stimulation frequencies. Rigorous design of ECC protocols is critical for effective use of strength loss as a readout in evaluating potential therapeutics for muscular dystrophy.

Rapid, redox-mediated mechanical susceptibility of the cortical microtubule lattice in skeletal muscle

“…ROS have been implicated in mediating rapid eccentric contraction-induced force loss in mdx muscle [ 19 , 28 ]. We pretreated mdx mice expressing either a mini- or micro-dystrophin with the non-specific antioxidant N -acetylcysteine (NAC) and assessed microtubule organization after eccentric contraction.…”

“…The microtubule lattice in transgenic mdx mice expressing truncated dystrophins was not affected by isometric contractions, which do not cause force loss in mdx muscle [ 17 ]. In contrast, passive lengthening of mdx muscle has been shown to induce mechanotransduction through the detyrosinated-tubulin microtubule lattice to stimulate heightened NOX2 ROS [ 12 , 13 ], although force loss does not result [ 19 ]. Here we found that passive muscle lengthening had no effect on microtubule organization in mdx mice expressing mini- or micro-dystrophin, which also presented with wildtype levels of detyrosinated tubulin [ 16 ].…”

“…We demonstrated here that a single dose of N -acetylcysteine administered to mdx mice expressing a mini- or micro-dystrophin prior to the in vivo eccentric contraction protocol was sufficient to completely protect against loss of transverse microtubules. N -acetylcysteine is a non-specific antioxidant [ 54 ] that has been shown to have a protective effect against force loss during mdx eccentric contractions [ 19 , 28 , 55 ] as well as effectively reduce ROS production in mdx skeletal muscle [ 55 ]. Due to its non-specific nature, NAC potentially diminishes ROS from multiple sources.…”

“…Microtubule disorganization in mdx muscle has also been associated with altered α-tubulin posttranslational modification [ 12 , 13 ], increased expression of the rare β-tubulin tubb6 during muscle regeneration [ 14 ], and altered production of reactive oxygen species (ROS) via NADPH oxidase 2 (NOX2) [ 15 ]. Several studies have shown that microtubule disorganization correlates with loss of muscle force production following eccentric (lengthening) contractions [ 9 , 11 , 16 ], which is one of the most robust and reproducible phenotypes of mdx mice [ [17] , [18] , [19] ]. Susceptibility to eccentric contraction is also linked to tubulin detyrosination [ 12 , 13 ].…”

Muscle weakness has a limited effect on motor control of gait in Duchenne muscular dystrophy

“…In individuals with DMD, the protein dystrophin is absent. This protein is expected to play an important role in ensuring the stability of the muscle cell membrane and protecting the muscles from contraction induced damage [ 4 – 6 ]. When the disease progresses, muscle damage will get the overhand and muscles will get infiltrated with non-contractile fibrofatty tissue [ 3 ].…”