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Quinn JL, Huynh T, Uaesoontrachoon K, Tatem K, Phadke A, Van der Meulen JH, Yu Q, Nagaraju K. Citation Quinn JL, Huynh T, Uaesoontrachoon K, Tatem K, Phadke A, Van der Meulen JH, Yu Q, Nagaraju K.
Research Center for Genetic Medicine, Children’s National Medical Center, Washington, DC, USA.
The Canberra Hospital, Endocrine Research Unit and Australian National University Medical School, Department of Paediatrics & Child Health, Canberra, Australia. Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington DC, USA; Research Center for Genetic Medicine, Children’s National Medical Center, Washington, DC, USA. Dystrophin deficiency causes contraction-induced injury and damage to the muscle fiber, resulting in sustained increase in intracellular calcium levels, activation of calcium-dependent proteases and cell death. Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder that affects 1 in 3500 boys in all populations 1. One of the major consequences of this calcium increase is the increased activation of calcium-dependent proteases that lead to protein degradation and eventually cell death 4.
In skeletal muscle, contraction is induced through a process of calcium-induced calcium release.
Dantrolene Sodium is an FDA approved skeletal muscle relaxant that has been safely administered to DMD patients without harmful side effects 11. We will use Dantrolene treatment to decrease intracellular calcium levels and thus decrease the amount of calcium-induced necrosis and improve the overall phenotype of the treated mice. After four weeks of treatment, forelimb and hindlimb GSMs were performed as previously described 13 but with an increase in the acclimation period from three to five consecutive days. After four weeks of treatment, voluntary locomotor activity was measured using an open-field digiscan apparatus (Omnitech Electronics, Columbus, Ohio) as previously described 13 except that the mice were acclimated for four days before data was collected. Prior to sacrifice, the extensor digitorium longus (EDL) muscles of the right hindlimbs were removed for comparison of in vitro force contractions between groups as previously described 14. After five weeks of treatment, quantification of inflammation in forelimb and hindlimb muscles of mice was achieved using live-animal optical imaging of Cathepsin-B (CTSB) enzyme activity using a caged near-infrared substrate (ProSense 680) as previously described 15.
The blood was collected by cardiac puncture immediately after euthanasia by carbon dioxide inhalation. Directly following euthanasia, the tibialis anterior (TA) muscles were removed and preserved in formalin. SERCA1 ATPase protein levels were measured in Gastrocnemius muscles from the mdx treatment and control groups.
RNA was isolated from frozen Gastrocnemius muscle using an integrated Trizol and RNeasy minikit (Qiagen, Valencia, CA) protocol with DNase I (Qiagen) treatment. For all proposed experiments and procedures, one-way ANOVA with Dunnett’s post-test comparison against a control was used for experiments involving more than two groups.
Bodyweight of each mouse was measured at four weeks of age and recorded every week of treatment up until sacrifice at ten weeks of age. Grip strength and open field testing for mdx and BL10 mice was performed after 4 weeks of treatment. Comparisons between BL10 and mdx Vehicle-treated mice as well as both mdx mice groups yielded no significant differences. We have previously shown that cathepsin activity in fore and hindlimbs is significantly increased in mdx mice 20. Cathepsin activity was measured from A) forelimb and B) hindlimb muscles as a quantification of inflammation. Hematoxylin and eosin (H&E) stained sections from the TA of each animal were analyzed in blinded fashion. No significant difference was observed in any of these measurements including A) specific muscle force, B) lengthening contractions to measure how the muscle responds to damage from over lengthening, or C) measuring the muscle’s ability to resist and recover from fatigue. To evaluate whether Dantrolene treatment altered SERCA1 expression, we did western blotting and densitometry normalized to ?-actin. Western blot and densitometry analysis performed on protein extracted from gastrocnemius muscle to measure SERCA1 protein levels in skeletal muscle.
In order to determine the effect of Dantrolene treatment on Ryr1 in skeletal muscle, qPCR to measure RyR1 levels was performed. In this study we demonstrated that Dantrolene treatment has no significant beneficial effects at the tested dose in young mdx mice.
Dantrolene is a known muscle relaxant and used for the treatment of malignant hyperthermia 11. Even though it has been shown previously that RyR1 is leaky in dystrophic muscle and contributes to the disease phenotype, and drugs that stabilize RyR1 are known to be effective in muscular dystrophy 23, we could not see a similar effect with Dantrolene in this experiment.
Dantrolene has previously been shown to enhance exon skipping in mdx mice but not improve muscle strength on its own 24. Lack of efficacy in our study could be a result of beginning treatment too late in the disease progression, at a point where significant damage had already occurred in the muscle fibers. Our analysis of the serum CK levels did show a significant decrease in the amount of CK in the blood. Additional assays that we performed on isolated EDL muscles indicate that not only strength, but also muscle recovery during in vitro fatigue and lengthening contraction protocols demonstrated no effect. But quickly people spotted the fact hot cross buns were not in any danger, and commenced mocking the EDL for the mix-up. Likewise, it sparked some angry responses before a deluge of people asking whether they’d bothered to actually read the article.
We will be provided with an authorization token (please note: passwords are not shared with us) and will sync your accounts for you. Skeletal muscle satellite cells (SCs) are Pax7+ myogenic stem cells that reside between the basal lamina and the plasmalemma of the myofiber.
Skeletal muscle is composed of multinucleated myofibers that are established during embryogenesis by fusion of myoblasts.
The FGFs are key players in the processes of proliferation and differentiation of a wide range of cells and tissues.
As the paracrine FGFs mediate their biological responses by binding to cell surface FGF receptors (FGFR1, FGFR2, FGFR3, FGFR4), FGFR impairment offers one possible approach for studying the effect of FGF2 signaling on SC performance.
If FGFR signaling is essential for regulating SC pool size, which in turn may be important for muscle homeostasis, then a better understanding of this topic is needed when considering future therapies for disease- or age-associated muscle wasting.
Experimental procedures were approved by the University of Washington Institutional Animal Care and Use Committee. RNA was isolated from freshly sorted myogenic and non-myogenic populations and reverse transcribed according to our published procedure (Day et al., 2010). It is known that the Ryanodine receptor (RyR1) on the sarcoplasmic reticular (SR) membrane controls calcium release.
It is caused by a mutation in the dystrophin gene, leading to the absence of the cytoskeletal protein dystrophin 2.
This increased degeneration from calcium-induced necrosis worsens the overall phenotype of DMD and hastens the loss of ambulation and eventual death due to respiratory and cardiac complications.
This occurs when the Ryanodine receptor (RyR1) on the sarcoplasmic reticular (SR) membrane interacts with voltage-dependent Ca2+ channels on the T-tubules such as dyhydropyridine receptors (DHPRs) and allows calcium release from the SR into the sarcoplasm when activated 8. This protein is responsible for pumping calcium out of the sarcoplasm and back into the SR following excitation 9. Dantrolene binds to the RyR1 receptor and inhibits the release of calcium from the SR during excitation-contraction by blocking its activation 12.
To test the efficacy of this treatment regime, we have performed a pre-clinical trial in mdx mice and will evaluate improvement using several criteria: functional, behavioral, histological, and molecular assays. Following acclimation, data was recorded over a 1-hour period each day for four consecutive days.
Briefly, mice were injected intraperitoneally with 1.5 nmol of ProSense 680 (VisEn Medical).
For histological analysis, muscles were embedded into paraffin blocks and subsequently sectioned and stained with haematoxylin and eosin (H&E) by Histoserv (Germantown, MD). Frozen muscle sections were homogenized as previously described 17 and equal amounts (50ug) were loaded and run in a reducing SDS-PAGE gel (Invitrogen, Grand Island, NY) at 150V. The two mdx treatment groups showed no significant change in body weight of the course of treatment after the mdx mice were broken up into groups based on normalizing bodyweight. Vehicle-treated mdx mice showed a reduction in absolute forelimb strength (7.88%) compared to BL10 mice.



There was no significant change in any measured parameters, including horizontal and vertical activity as well as movement and rest time, between any of the groups (Table 1).
Comparisons of ten standard parameters yielded no significant change between the two mdx groups. The EDL muscles of mdx mice had a significant decrease (31.98%) in specific force relative to BL10 mice. In untreated mdx mice, there was an 11.76 fold increase in creatine kinase in serum collected from mice prior to sacrifice relative to wild type controls. Dantrolene failed to improve the muscle phenotype as measured by functional, behavioral, histological, and imaging assays.
It decreases the calcium released in skeletal muscle upon stimulation, resulting in decreased muscle contraction 19. The reasons for this discrepancy are not clear, but could be due to the muscle relaxant properties of the drug. Our study confirms their findings despite using a higher dose of the drug to treat older mdx mice for a longer period of time (8 weeks).
Previous studies have demonstrated that calpains, calcium-dependent cytosine proteases, are present at elevated levels in mdx muscle by 4 weeks of age 25. This calcium ATPase is found on the SR membrane in muscle fibers, and is responsible for pumping calcium out of the sarcoplasm and back into the SR. Additionally, the histological scoring showed no significant change in any of the eight measured parameters, indicating existing damage of the fibers was not altered.
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This page doesn't support Internet Explorer 6, 7 and 8.Please upgrade your browser or activate Google Chrome Frame to improve your experience. In mature muscles, SCs are typically quiescent, but can be activated in response to muscle injury. In accordance with our findings, a recent study reported that FGF2 is required to remove age-associated proliferative inhibition of SCs (Li et al., 2014). Gaining further understanding of the role of the FGFR system in myogenesis requires models that facilitate direct FGFR ablation, bypassing downstream interventions that may not specifically target individual FGFRs and may affect additional tyrosine kinase receptor cascades. The R26mTmG reporter operates on a membrane-localized dual fluorescent system where all cells express Tomato until Cre-mediated excision of the Tomato gene allows for GFP expression in the targeted cell lineage (Muzumdar et al., 2007). Dantrolene, an FDA approved skeletal muscle relaxant, inhibits the release of calcium from the SR during excitation-contraction and suppresses uncontrolled calcium release by directly acting on the RyR complex to limit its activation.
This complex is activated by signals proliferated along the T-tubules and leads to muscle contraction. It has been demonstrated that this protein is dysfunctional in mdx mice and further contributes to increased levels of sarcoplasmic calcium 10.
All handling and experimentation with mice was conducted in accordance with our IACUC guidelines under approved protocols.
The gel was then blotted transferred onto PVDF membranes and incubated overnight with anti-SERCA1 ATPase monoclonal antibody (1:2500, Thermo Scientific, Waltham, MA) at 4°C.
The Taqman Gene Expression Assays Hprt (Mm01545399_m1) and Ryr1 (Mm01175211_m1) were used for analysis. This resulted in a significant difference (P-value = 0.0115) between the bodyweights of the BL10 group and the mdx groups starting at two weeks of treatment and continuing until the end of the trial.
Treatment of mdx mice with Dantrolene resulted in a 16.69% reduction in strength compared to the untreated mdx mice (Fig. Untreated mdx mice showed a significant (84.87%) increase in cathepsin activity in the forelimbs using optical imaging compared to BL10 mice. Treatment with Dantrolene resulted in a significant decrease (76.42%) in serum CK levels compared to untreated mdx mice (Fig. Dantrolene-treated mice showed a significant decrease (33%) in SERCA1 levels compared to vehicle-treated mdx mice (Fig. We did not find differences in RyR1 expression between the two mdx mouse groups (data not shown). In fact, functional assessments such as grip strength and open field activity were decreased in Dantrolene-treated mice compared to their vehicle-treated counterparts. These findings were confirmed by analysis of H&E sections that also indicate no change in the histological profile, including inflammation, between the two groups.
In addition, we conducted additional functional assays to determine the effect of the drug. This means that calcium-induced necrosis had already initiated by the time we started treatment. Based on the histology and functional assays, it is unlikely that this change is based on improved muscle health. We expected that Dantrolene treatment and the resulting intracellular calcium decrease would result in an increase in SERCA1 levels in muscle fibers based on previous research with rat hearts 27.
Our data suggests that during early necrosis when there is cycles of damage, regeneration, and re-injury; administration of Dantrolene is ineffective. Depending on the magnitude of tissue trauma, SCs may divide minimally to repair subtle damage within individual myofibers or produce a larger progeny pool that forms new myofibers in cases of overt muscle injury. Proliferating progeny maintain Pax7 expression as their quiescent progenitors, but distinctly, are also MyoD-positive (Zammit et al., 2004). In the current study we have aimed to gain insight into the role of FGFR1 during adult myogenesis using Fgfr1-ablated mice.
This study examines whether Dantrolene can reduce the disease phenotype in the mdx mouse model of muscular dystrophy. Without this anchoring protein, the muscle’s structure is compromised which in turn results in contraction-induced injury. In fact, restoring calcium homeostasis by increasing calcium channels or the use of drugs that increase cellular proteins that stabilize calcium channels has been shown to improve muscle function and strength 5,6,7. At optimal muscle length, the force developed was measured during trains of stimulation (300 milliseconds) with increasing frequencies until the highest plateau was achieved. After washing, membranes were probed with HRP-conjugated anti-mouse IgG1 (1:2000, Invitrogen) and incubated with ECL Western Blotting Substrate (GE Healthcare, Piscataway, NJ) and processed on X-ray film (Denville Scientific, South Plainfield, NJ). There was no significant difference in bodyweight over time between the two mdx groups (Fig.
It is unlikely that improved calcium handling in previously damaged fibers would reverse the damage, but only slow further deterioration. Instead, it is highly likely that this decrease in serum CK is a result of decreased activity. We performed Western blotting on gastrocnemius muscles and found a 33% decrease in SERCA1 protein levels compared to mdx vehicle-treated mice.
Therefore, we conclude that Dantrolene alone at this dose does not improve the overall phenotype of the mdx mouse. SC transition through proliferation, differentiation and renewal is governed by the molecular blueprint of the cells as well as by the extracellular milieu at the SC niche. Using this muscle-specific reporter model, the isolated cells are sorted into myogenic and non-myogenic populations according to GFP vs.
This repeated injury leads to cycles of inflammation and muscle degeneration, which in turn leads to muscle weakening and loss of function. Because we found significant differences in bodyweight between BL10 and mdx mice, we also normalized strength to body weight.
Mice treated with Dantrolene showed no significant decrease (18.17%) in inflammation in the forelimbs compared to untreated mdx mice. Additionally, mdx mice were significantly less resistant to damage occurring from lengthening compared to BL10 mice (Fig. Additionally, it is quite possible that a longer duration of treatment is necessary to observe any benefit from the drug in mdx mice. As a muscle relaxant, Dantrolene slowed down the breathing rate and reduced activity in the mice 19, reducing CK levels in the blood.
We suspect this difference in effect is likely due to the different muscle type, dosage, duration, and model. In particular, the role of the fibroblast growth factor (FGF) family in regulating SCs during growth and aging is well recognized.


Depending on the magnitude of tissue trauma, SCs may divide minimally to repair subtle damage within individual myofibers or produce a larger progeny pool that forms new myofibers in cases of overt muscle injury (Grounds and Yablonka-Reuveni, 1993; Hawke and Garry, 2001).
However, it has been reported that excess FGF2 harbored in the myofibers of aging mice leads to SC depletion due to detrimental proliferation (without self-renewal), rather than retention of the quiescent state (Chakkalakal et al., 2012). Tomato fluorochrome, respectively, combined with antigen-based sorting for maximal purification as we previously described (Stuelsatz et al., 2014). Raw qPCR cycle threshold values for each individual sample were normalized to eukaryotic translation elongation factor 2 (Eef2) reference gene expression as in (Phelps et al., 2013).
Transcript expression analysis was done by semi-quantitative RT-PCR according to our standard protocol (Day et al., 2007).
We found that treatment with Dantrolene resulted in decreased grip strength and open field behavioral activity in mdx mice. The repeated damage to the muscle fiber due to contraction also results in transient tears in the sarcolemma of dystrophic muscles, thereby allowing the influx of extracellular calcium 3. Functional testing was conducted at 8 weeks of age followed by optical imaging the next week.
Additionally, the muscle was subjected to a protocol of ten lengthening contractions separated by one minute rest intervals.
Untreated mdx mice showed a significant decrease (24.23%) in normalized forelimb strength relative to the BL10 mice. To minimize variation in the functional and behavioral assays, only female mice were used because there are clear gender differences in mdx muscle pathology between sexes 21 and to reduce variability others and we have suggested that pre-clinical tests should be performed on groups of mdx mice of the same sex 22. This assay however, establishes that Dantrolene did affect calcium handling within mdx muscle fibers but it did not result in an improved disease phenotype.
Of the several FGFs shown to affect SCs, FGF1, FGF2, and FGF6 proteins have been documented in adult skeletal muscle. Hence, means for direct ablation of FGF2 signaling are needed to assist in determining its role in SC performance during aging. MyoD is well recognized as a master regulator of the myogenic lineage specification during embryogenesis (Weintraub et al., 1991). Nevertheless, as discussed in the Introduction, FGFR2 has been considered not relevant in adult myogenesis and indeed, as shown in Results, Fgfr2 transcript expression in SCs and their progeny is negligible. There was no significant difference in inflammation either by optical imaging analysis of cathepsin activity or histological (H&E) analysis. This elevated influx of calcium results in a sustained increase in intracellular calcium levels.
At 10 weeks of age the mice were sacrificed and tissue samples were immediately collected, weighed, snap frozen in liquid nitrogen-cooled isopentane, and stored at -80?C for later processing. Mdx mice treated with Dantrolene showed a 13.58% decrease compared to the untreated mdx group (Fig. Treatment with Dantrolene showed no improvement in muscle force generated after lengthening contractions (Fig.
These prototypic paracrine FGFs transmit their mitogenic effect through the FGFRs, which are transmembrane tyrosine kinase receptors.
While our overexpression studies have suggested different modes of function for FGFR1 and FGFR4 (Kwiatkowski et al., 2008), it is unknown whether these two FGFRs can compensate for each other during SC myogenesis.
Cell sorting was then performed using an Influx Cell Sorter (BD Biosciences) equipped with 350, 488, and 638 nm lasers. Genes were considered expressed if cycle threshold values (raw Ct) of less than 33 cycles were detected.
In vitro force contraction measures showed no changes in EDL muscle-specific force, lengthening-contraction force deficit, or fatigue resistance.
Finally, the muscle was subjected to a fatigue protocol consisting of 60 isometric contractions for 300 milliseconds each, once every 5 seconds.
Using the mouse model, we show here that of the four Fgfr genes, only Fgfr1 and Fgfr4 are expressed at relatively high levels in quiescent SCs and their proliferating progeny. Thereby, MyoDCre-mediated excision of floxed genes would occur in the embryonic muscle and be stably maintained in the myogenic lineage through adult life. Expression of Tbp (TATA box binding protein) housekeeping control gene served as quality and loading control as in (Stuelsatz et al., 2012). Hindlimb measurements were unable to ascertain a difference between mdx and BL10 mice even after normalization (data not shown). Finally, no significant differences were observed between any of the groups in muscle fatigue or recovery (Fig. However, the inhibitory drug used, SU5402 (Mohammadi et al., 1997), can theoretically target all FGFRs based on its effect on blocking FGFR tyrosine kinase function. Here we show that myogenic-specific ablation of Fgfr1 does not appear to influence muscle morphology or regeneration following cardiotoxin-induced damage in adult mice.
The FGFR2fl allele contains loxP sites flanking exons 8-10 that encode a portion of the ligand binding Ig-3 domain and the transmembrane domain (Yu et al., 2003).
Further, Dantrolene-treated mice showed decreased SERCA1 but not RyR1 expression in skeletal muscle. The force was recorded every minute during the repetitive contractions and again five minutes after to measure recovery. Neither muscle histology nor muscle regeneration following cardiotoxin-induced injury were overtly affected in Fgfr1-ablated mice. Nevertheless, our study provides novel evidence for the obligatory role for FGFR1 in mediating FGF2 mitogenic effect on SCs that is not compensated by FGFR4, which is also highly expressed in SCs.
Data was acquired at 20,000–100,000 events per sample and sorted cells were collected in our culture media described below.
These results suggest that Dantrolene treatment alone has no significant beneficial effects at the tested doses in young mdx mice. Myogenic-specific ablation or overexpression of Spry1, a member of the Sprouty family of negative regulators of receptor tyrosine kinase signaling (Cabrita and Christofori, 2008), were also employed to modulate FGF signaling during adult myogenesis (Chakkalakal et al., 2012). However, the SC mitogenic response to FGF2 is drastically repressed in isolated myofibers prepared from Fgfr1-ablated mice. Collectively, our study indicates that FGFR1 is important for FGF-mediated proliferation of SCs and its mitogenic role is not compensated by FGFR4 that is also highly expressed in SCs. Increased protein degradation results from elevated free calcium levels found in muscle from mdx mice.
Overexpression of SERCA1a in the mdx diaphragm reduces susceptibility to contraction-induced damage. Leaky ryanodine receptors in ?-sarcoglycan deficient mice: a potential common defect in muscular dystrophy.
Preclinical drug trials in the mdx mouse: assessment of reliable and sensitive outcome measures.
Non-invasive optical imaging of muscle pathology in mdx mice using cathepsin caged near-infrared imaging. Evaluation of skeletal and cardiac muscle function after chronic administration of thymosin beta-4 in the dystrophin deficient mouse. Identification of disease specific pathways using in vivo SILAC proteomics in dystrophin deficient mdx mouse. Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle. Gender dimorphism influences extracellular matrix expression and regeneration of muscular tissue in mdx dystrophic mice. Towards developing standard operating procedures for pre-clinical testing in the mdx mouse model of Duchenne muscular dystrophy. Dantrolene enhances antisense-mediated exon skipping in human and mouse models of Duchenne muscular dystrophy.
Dantrolene: effects on abnormal intracellular Ca(2+) handling and inotropy in postinfarcted rat myocardium.



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