Abstract
BackgroundDystrophin 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 ECC-induced 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.MethodsWe 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).ResultsThe 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.ConclusionsThese 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.
Highlights
Mutation in the Duchenne muscular dystrophy (DMD) gene can detrimentally affect the expression and function of its product dystrophin [1], a protein that stabilizes the sarcolemma during contraction by linking the extracellular matrix to the intracellular cytoskeleton [2]
Mechanical factors of the eccentric contraction (ECC) that cause strength loss in wild type (WT) skeletal muscle include work performed by the muscle [13], initial length of the muscle, amplitude of the length change [14], and maximal muscle tension [15] during the ECC, with the latter being measured as the ratio of maximal eccentric to isometric force (ECC:ISO)
Analyzing the effect of contraction velocity and duration within given length changes showed that ECCs with slower velocities and longer durations resulted in greater loss of isometric force compared to fast and short ECCs (Fig. 1e)
Summary
Mutation in the DMD gene can detrimentally affect the expression and function of its product dystrophin [1], a protein that stabilizes the sarcolemma during contraction by linking the extracellular matrix to the intracellular cytoskeleton [2]. Lack of dystrophin renders skeletal muscle susceptible to injury [3], eccentric contraction (ECC)-induced strength loss [4,5,6]. Such loss of strength in the mdx mouse model of Duchenne muscular dystrophy (DMD) is associated with cytosolic calcium influx [7], generation of reactive oxygen species (ROS) [8] and disruption of the mechanisms responsible for activating, generating, and transmitting force.
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