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Muscle Twitch Kinetics Are Dependent on Muscle Group, Disease State, and Age in Duchenne Muscular Dystrophy Mouse Models.

ABSTRACT: Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by the lack of functional dystrophin protein. In muscular dystrophy preclinical research, it is pertinent to analyze the force of the muscles affected by the disease to assess pathology and potential effectiveness of therapeutic interventions. Although muscles function at sub-maximal levels in vivo, maximal tetanic contractions are most commonly used to assess and report muscle function in muscular dystrophy studies. At submaximal activation, the kinetics of contraction and relaxation are heavily impacted by the kinetics of the single twitch. However, maximal tetanic force is often the main, if not sole, outcome measured in most studies, while contractile kinetics are rarely reported. To investigate the effect of muscle disease on twitch contraction kinetics, isolated diaphragm and extensor digitorum longus (EDL) muscles of 10-, 20-week, "het" (dystrophin deficient and utrophin haplo-insufficient), and 52-week mdx (dystrophin deficient) mice were analyzed and compared to wild-type controls. We observed that twitch contractile kinetics are dependent on muscle type, age, and disease state. Specific findings include that diaphragm from wildtype mice has a greater time to 50% relaxation (RT50) than time to peak tension (TTP) compared to the het and mdx dystrophic models, where there is a similar TTP compared to RT50. Diaphragm twitch kinetics remain virtually unchanged with age, while the EDL from het and mdx mice initially has a greater RT50 than TTP, but the TTP increases with age. The difference between EDL contractile kinetics of dystrophic and wildtype mice is more prominent at young age. Differences in kinetics yielded greater statistical significance compared to previously published force measurements, thus, using kinetics as an outcome parameter could potentially allow for use of smaller experimental groups in future study designs. Although this study focused on DMD models, our findings may be applicable to other skeletal muscle conditions and diseases.

SUBMITTER: Peczkowski KK

PROVIDER: S-EPMC7545010 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMuscle wasting occurs in response to various physiological and pathological conditions, including ageing and Duchenne muscular dystrophy (DMD). Transforming growth factor-β1 (TGF-β1) contributes to muscle pathogenesis in elderly people and DMD patients; inhibition of TGF-β1 signalling is a promising therapeutic strategy for muscle-wasting disorders. Hemojuvelin (HJV or Hjv as the murine homologue) is a membrane-bound protein that is highly expressed in skeletal muscle, heart, and liver. In hepatic cells, Hjv acts as a coreceptor for bone morphogenetic protein, a TGF-β subfamily member. The aim of this study was to investigate whether Hjv plays an essential role in muscle physiological and pathophysiological processes by acting as a coreceptor for TGF-β1 signalling.MethodsConventional and conditional Hjv knockout mice as well as mdx and aged mice transfected with Hjv overexpression vector were used to study the role of Hjv in muscle physiology and pathophysiology. qRT-PCR, western blotting, and immunohistochemistry examinations were conducted to evaluate gene, protein, and structural changes in vivo and in vitro. Exercise endurance was determined using treadmill running test, and muscle force was detected by an isometric transducer. RNA interference, immunoprecipitation, and dual-luciferase reporter assays were utilized to explore the mechanism by which Hjv regulates TGF-β1 signalling in skeletal muscle.ResultsConventional and conditional Hjv knockout mice displayed muscle atrophy, fibrosis, reduced running endurance, and muscle force. HJV was significantly down-regulated in the muscles of DMD patients (n = 3, mean age: 11.7 ± 5.7 years) and mdx mice as well as in those of aged humans (n = 10, 20% women, mean age: 75.1 ± 9.5 years) and mice. Overexpression of Hjv rescued dystrophic and age-related muscle wasting. Unlike its function in hepatic cells, the bone morphogenetic protein downstream phosphorylated p-Smad1/5/8 signalling pathway was unchanged, but TGF-β1, TGF-β receptor II (TβRII), and p-Smad2/3 expression were increased in Hjv-deficient muscles. Mechanistically, loss of Hjv promoted activation of Smad3 signalling induced by TGF-β1, whereas Hjv overexpression inhibited TGF-β1/Smad3 signalling by directly interacting with TβRII on the muscle membrane.ConclusionsOur findings identify an unrecognized role of HJV in skeletal muscle by regulating TGF-β1/Smad3 signalling as a coreceptor for TβRII. Unlike the TGF-β1/Smad3 pathway, HJV could be a reliable drug target as its expression is not widespread. Novel therapeutic strategies could potentially be devised to interfere only with the muscle function of HJV to treat DMD and age-related muscle wasting.

Project description:BackgroundDuchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy without an effective treatment, caused by mutations in the DMD gene, leading to the absence of dystrophin. DMD results in muscle weakness, loss of ambulation, and death at an early age. Metabolomics studies in mdx mice, the most used model for DMD, reveal changes in metabolites associated with muscle degeneration and aging. In DMD, the tongue muscles exhibit unique behavior, initially showing partial protection against inflammation but later experiencing fibrosis and loss of muscle fibers. Certain metabolites and proteins, like TNF-α and TGF-β, are potential biomarkers for dystrophic muscle characterization.MethodsTo investigate disease progression and aging, we utilized young (1 month old) and old (21-25 months old) mdx and wild-type tongue muscles. Metabolite changes were analyzed using 1H nuclear magnetic resonance, while TNF-α and TGF-β were assessed using Western blotting to examine inflammation and fibrosis. Morphometric analysis was conducted to assess the extent of myofiber damage between groups.ResultsThe histological analysis of the mid-belly tongue showed no differences between groups. No differences were found between the concentrations of metabolites from wild-type or mdx whole tongues of the same age. The metabolites alanine, methionine, and 3-methylhistidine were higher, and taurine and glycerol were lower in young tongues in both wild type and mdx (p < 0.001). The metabolites glycine (p < 0.001) and glutamic acid (p = 0.0018) were different only in the mdx groups, being higher in young mdx mice. Acetic acid, phosphocreatine, isoleucine, succinic acid, creatine, and the proteins TNF-α and TGF-β had no difference in the analysis between groups (p > 0.05).ConclusionsSurprisingly, histological, metabolite, and protein analysis reveal that the tongue of old mdx remains partially spared from the severe myonecrosis observed in other muscles. The metabolites alanine, methionine, 3-methylhistidine, taurine, and glycerol may be effective for specific assessments, although their use for disease progression monitoring should be cautious due to age-related changes in the tongue muscle. Acetic acid, phosphocreatine, isoleucine, succinate, creatine, TNF-α, and TGF-β do not vary with aging and remain constant in spared muscles, suggesting their potential as specific biomarkers for DMD progression independent of aging.

Project description:Albeit increased serum CK level and abnormal muscle histology are always present, boys with DMD are phenotipically indistinguishable from the normal ones at birth and, in their first years of life, acquire early motor milestones at normal times. A clear defect in muscle function becomes generally apparent by the end of the second year. As the disease is typically diagnosed between the ages of 3 and 7, the first two years are often considered and referred to as clinically presymptomatic. As a defined gene expression signature was shown to characterize these symptomatic patients we sought to investigate whether and to which extent alterations may be also present in muscle from presymptomatic DMD infants. To this aim, we used the Affymetrix technology to compare the individual expression profiles of 19 DMD patients with age at biopsy scattered along the first two years of the disease with those of 14 age matched controls. This approach allowed us to describe with high resolution the altered transcriptional state that characterizes this early, presymptomatic phase of the disease and highlight some molecular pathways as potential critical targets in the pathophysiology of the disease. Experiment Overall Design: -Subjects Experiment Overall Design: Relevant data about the participants are posted under supplementary material (Table ST1). All DMD patients (n=22) had diagnosis of Duchenne Muscular Dystrophy based on the absence of dystrophin immunoreactivity on quadriceps muscle sections. None of the participants at the time of biopsy was or had been under corticosteroid treatment. Control biopsies (n=14) were selected from diagnostic specimens which were not given a diagnosis of neuromuscular disease nor showed any non-specific myopathic signs. A MIAME compliant description of this study is posted under supplementary Material (MIAME compliance). Experiment Overall Design: -RNA extraction. Experiment Overall Design: Total RNA was extracted from frozen quadriceps muscle biopsies by TriZol. (TriZol reagent, Invitrogen). RNA was further purified using the RNAeasy mini kit following the RNA cleanup protocol as indicated by the manufacturer (QIAGEN). RNA purity and integrity was assessed by spectophotometric analysis and agarose gel electrophoresis. Experiment Overall Design: -Affymetrix Genechips. Experiment Overall Design: In this work we made use of one chip type: the affymetrix HG-U133A genechipCRNA synthesis was performed using 5ug of total RNA as template, as described in the Affymetrix Gene Expression Manual. Genechips were washed and stained in an Affymetrix fluidic station 430. To avoid overcorrelation, samples were processed 8 at the time and arranged to have both DMD and control samples in each experimental session.

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Muscle Twitch Kinetics Are Dependent on Muscle Group, Disease State, and Age in Duchenne Muscular Dystrophy Mouse Models.

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