We performed a placebo-controlled pre-clinical study to determine if sodium 4-phenylbutyrate (4PB) can reduce contraction-induced myofiber damage in the mdx mouse model of Duchenne muscular dystrophy (DMD). At 72 h post-eccentric contractions, 4PB significantly increased contractile torque and reduced myofiber damage and macrophage infiltration. We conclude that 4PB, which is approved by Health Canada (Pheburane) and the United States Food and Drug Administration (Buphenyl) for urea cycle disorders, might modify disease severity in patients with DMD.
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.
We report that, labeling mouse muscle tissue, with mouse monoclonal antibodies specific to slow or fast myosin heavy chain (sMyHC and fMyHC, respectively), can lead to artefactual labeling of damaged muscle fibers, as hybrid fibers (sMyHC+ and fMyHC+). We demonstrate that, such erroneous immunophenotyping of muscle may be avoided, by performing colabeling or serialsection- labeling, to identify damaged fibers. The quadriceps femorismuscle group (QF) in 7-month-old, male, C57BL/6J mice had: 1.21±0.21%, 98.34±1.06%, 0.07±0.01%, and 0.53±0.85% fibers, that were, sMyHC+, fMyHC+, hybrid, and damaged, respectively. All fibers in the tibialis anterior muscle (TA) of 3-month-old, male, C57BL/6J mice were fMyHC+; and at 3 days after injurious eccentric contractions, there was no fiber-type shift, but ~ 18% fibers were damaged.
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.
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.
BACKGROUNDMinimally Invasive Muscle Embedding (MIME), is a technique developed in our laboratory, to facilitate the development of donor‐cell‐derived muscle fibers in a host muscle. MIME involves passing a sterile needle through a host muscle, and then implanting a segment of donor muscle tissue along with its myogenic satellite cells, in the needle track within the host muscle.METHODSWe validated MIME, by implanting a single extensor digitorum longus (EDL) muscle, from donor mice (N=2), which expressed a red fluorescent protein (RFP), into the left tibialis anterior muscle (TA), of immunodeficient host mice (N=4), which expressed a green fluorescent protein (GFP). In the same host mice, we performed a SHAM procedure on the right TA. In another set of host mice, we implanted by MIME, segments of human cadaveric TA (3 days post‐mortem), or performed a SHAM procedure (N=4 per group). We triggered concerted muscle degeneration and regeneration by injecting 1.2% barium chloride (BACL) into the TAs after MIME or SHAM. We collected TAs at 14 days after implanting RFP+ mouse donor muscle, and at 12 weeks after implanting human donor muscle. We performed histological and physiological assays to compare MIME‐ and SHAM‐treated host TA muscles. We studied histological images with Image J software to obtain quantitative data. We performed statistical analyses (ANOVAs) with SigmaStat or SPSS software.RESULTSIn host TAs implanted with RFP+ mouse EDL, 22±4% and 78±4% muscle fibers were RFP+ and GFP+, respectively. There were no RFP+ fibers in SHAM‐treated muscles. All RFP+ fibers were positive for desmin and dystrophin, and 65±4 % fibers were centrally nucleated. Donor‐derived fibers had multiple sarcomeres and nuclear domains in series. In host TAs implanted with human muscle, ~30% of the host muscle was of donor origin (GFP−), and the majority of GFP− fibers were positive for human‐specific markers. The contractile torque produced by MIME and SHAM TAs was not statistically different at 12 weeks post‐MIME, and there was no statistical difference compared to torque produced before MIME or SHAM procedures. Data are reported as Mean±SEM. P<0.05 considered significant.CONCLUSIONThrough MIME, we confirm that, human cadaveric muscle, implanted up to 3 days post‐mortem, can generate donor‐derived muscle fibers that are morphologically and functionally healthy. Our studies are translationally‐relevant; since MIME can be adapted to implant myogenic tissue of human cadaveric origin, to generate donor‐derived muscle fibers in human recipients (example: patients with muscle loss from muscle disease, trauma, or aging), with suitable immunosuppressive and rehabilitative therapies.Support or Funding InformationFunded by a Pilot Grant from the Alliance for Regenerative Rehabilitation Research and Training (AR3T), NIH 1R03HD091648‐01 and 5R03HD091648‐02 from NICHD, and a Faculty Startup Package and FRAP award from Wayne State University, to JAR. De‐identified, cadaveric human muscle tissue, was made available through the Body Bequest Program at Wayne State University. MIME facilitates donorderived myogenesisimageMIME facilitates donorderived myogenesisThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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