μ-Crystallin, encoded by the CRYM gene, binds the thyroid hormones, T 3 and T 4 . Because T 3 and T 4 are potent regulators of metabolism and gene expression, and CRYM levels in human skeletal muscle can vary widely, we investigated the effects of overexpression of Crym . We generated transgenic mice, Crym tg, that expressed Crym protein specifically in skeletal muscle at levels 2.6–147.5 fold higher than in controls. Muscular functions, Ca 2+ transients, contractile force, fatigue, running on treadmills or wheels, were not significantly altered, although T 3 levels in tibialis anterior (TA) muscle were elevated ~190-fold and serum T 4 was decreased 1.2-fold. Serum T 3 and thyroid stimulating hormone (TSH) levels were unaffected. Crym transgenic mice studied in metabolic chambers showed a significant decrease in the respiratory exchange ratio (RER) corresponding to a 13.7% increase in fat utilization as an energy source compared to controls. Female but not male Crym tg mice gained weight more rapidly than controls when fed high fat or high simple carbohydrate diets. Although labeling for myosin heavy chains showed no fiber type differences in TA or soleus muscles, application of machine learning algorithms revealed small but significant morphological differences between Crym tg and control soleus fibers. RNA-seq and gene ontology enrichment analysis showed a significant shift towards genes associated with slower muscle function and its metabolic correlate, β-oxidation. Protein expression showed a similar shift, though with little overlap. Our study shows that μ-crystallin plays an important role in determining substrate utilization in mammalian muscle and that high levels of μ-crystallin are associated with a shift toward greater fat metabolism.

Ibuprofen, a nonsteroidal anti-inflammatory drug, and nitric oxide (NO) donors have been reported to reduce the severity of muscular dystrophies in mice associated with the absence of dystrophin or -sarcoglycan, but their effects on mice that are dystrophic due to the absence of dysferlin have not been examined. We have tested ibuprofen, as well as isosorbide dinitrate (ISDN), a NO donor, to learn whether used alone or together they protect dysferlin-null muscle in A/J mice from large strain injury (LSI) induced by a series of high strain lengthening contractions. Mice were maintained on chow containing ibuprofen and ISDN for 4 weeks. They were then subjected to LSI and maintained on the drugs for 3 additional days. We measured loss of torque immediately following injury and at day 3 postinjury, fiber necrosis, and macrophage infiltration at day 3 postinjury, and serum levels of the drugs at the time of euthanasia. Loss of torque immediately after injury was not altered by the drugs. However, the torque on day 3 postinjury significantly decreased as a function of ibuprofen concentration in the serum (range, 0.67-8.2g/ml), independent of ISDN. The effects of ISDN on torque loss at day 3 postinjury were not significant. In long-term studies of dysferlinopathic BlAJ mice, lower doses of ibuprofen had no effects on muscle morphology, but reduced treadmill running by 40%. Our results indicate that ibuprofen can have deleterious effects on dysferlin-null muscle and suggest that its use at pharmacological doses should be avoided by individuals with dysferlinopathies.

IntroductionDysferlin‐deficient murine muscle sustains severe damage after repeated eccentric contractions.MethodsWith a robotic dynamometer, we studied the response of dysferlin‐sufficient and dysferlin‐deficient mice to 12 weeks of concentrically or eccentrically biased contractions. We also studied whether concentric contractions before or after eccentric contractions reduced muscle damage in dysferlin‐deficient mice.ResultsAfter 12 weeks of concentric training, there was no net gain in contractile force in dysferlin‐sufficient or dysferlin‐deficient mice, whereas eccentric training produced a net gain in force in both mouse strains. However, eccentric training induced more muscle damage in dysferlin‐deficient vs dysferlin‐sufficient mice. Although concentric training produced minimal muscle damage in dysferlin‐deficient mice, it still led to a prominent increase in centrally nucleated fibers. Previous exposure to concentric contractions conferred slight protection on dysferlin‐deficient muscle against damage from subsequent injurious eccentric contractions.DiscussionConcentric contractions may help dysferlin‐deficient muscle derive the benefits of exercise without inducing damage.

B6.A‐Dysf prmd/GeneJ (BLAJ) mice model human limb‐girdle muscular dystrophy 2B (LGMD2B), which is linked to mutations in the dysferlin (DYSF) gene. We tested the hypothesis that, the calcium ion (Ca2+) channel blocker diltiazem (DTZ), reduces contraction‐induced skeletal muscle damage, in BLAJ mice. We randomly assigned mice (N = 12; 3–4 month old males) to one of two groups – DTZ (N = 6) or vehicle (VEH, distilled water, N = 6). We conditioned mice with either DTZ or VEH for 1 week, after which, their tibialis anterior (TA) muscles were tested for contractile torque and susceptibility to injury from forced eccentric contractions. We continued dosing with DTZ or VEH for 3 days following eccentric contractions, and then studied torque recovery and muscle damage. We analyzed contractile torque before eccentric contractions, immediately after eccentric contractions, and at 3 days after eccentric contractions; and counted damaged fibers in the injured and uninjured TA muscles. We found that DTZ improved contractile torque before and immediately after forced eccentric contractions, but did not reduce delayed‐onset muscle damage that was observed at 3 days after eccentric contractions.

BackgroundThe L‐type Ca2+ channel blocker diltiazem (DTZ) protects dysferlin‐null A/J mice from muscle damage induced by large‐strain eccentric contractions (20 repetitions) (1). One of the disadvantages of studying the A/J mouse strain is that, it does not have a true control mouse strain. Additionally, A/J mice carry several non‐dysferlin mutations that might have an effect on muscle function (2). BLAJ mice, which carry the same dysferlin mutation as A/J mice, albeit in the C57BL/6J background, show lesser muscle damage than A/J mice after large‐strain eccentric contractions (3). We therefore developed a new model of eccentric exercise (40 repetitions of medium‐strain eccentric contractions) that reliably induces sufficient damage in BLAJ mouse muscle, in order to test if DTZ blocks contraction‐induced muscle damage in BLAJ mice.MethodsWe studied the tibialis anterior (TA) muscle in 3–4 month old, male, dysferlin‐null BLAJ mice. We measured contractile torque and exposed the TA muscle to eccentric contractions (one bout; 40 repetitions; 90–160 □ plantarflexion superimposed on maximal tetany of ankle dorsiflexors) with a custom‐built dynamometer. For one group of mice (Vehichle, VEH, N = 6 mice), we gave intraperitoneal injections of distilled water, once daily, for one week prior to eccentric contractions and three days thereafter (10 ul per gram body weight). For another group of mice (DTZ, N = 6 mice), we gave intraperitoneal injections of DTZ (72 mg/kg/day), once daily, following a similar injection schedule as the VEH group (we dissolved 72 mg DTZ in 10 ml distilled water and injected 10 ul per gram body weight). We assessed baseline contractile torque, torque changes after eccentric contractions, and histology of unexercised and exercised TA muscles.ResultsData are summarized in Table 1. DTZ improved Peak Twitch Torque, but did not provide protection against muscle damage from eccentric contractions.ConclusionDiltiazem might not offer protection against all types of contraction‐induced damage to dysferlin‐null muscle.Translational RelevanceDiltiazem might not be able to protect muscles in patients with dysferlin deficiency if muscle damage exceeds a certain threshold level.Support or Funding InformationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.