Kristine M. Wadosky1, Jessica E. Rodriguez1, Luge Li1, Dan Bogan1, Joe N Kornegay1 and Monte S Willis1,2
1 Pathology & Laboratory Medicine
2 McAllister Heart Institute, University of North Carolina, Chapel
Hill, NC
Duchenne muscular dystrophy (DMD) is an incurable and lethal genetic disorder characterized by progressive muscle wasting caused by the loss of functional dystrophin. Activation of both calpain and the proteasome degradation systems has been reported in the pathogenesis of DMD in skeletal muscle. In fact, calpain and proteasome inhibitors have been used as therapies in mouse models. Here we investigated specific calpain and proteasome activities of 5 skeletal muscles and the left ventricle (LV) in a more severe canine model of DMD (golden retriever muscular dystrophy [GRMD]). The expression of ubiquitin proteasome system components and calpain 1 and 2 were increased in GRMD skeletal muscle generally, but decreased in the LV. We identified significant increases in all three proteasome activities (trypsin-, chymotrypsin-, and caspase-like) in only 1 GRMD muscle and no activation of any proteasome activities in the LV. Calpain 1&2 activities were increased in 2 GRMD skeletal muscles and the LV compared to controls. Furthermore, protease activity of most GRMD skeletal muscles studied correlate with the degree of atrophy and hypertrophy found in the individual muscles. As proteasome and calpain inhibitors continue to be considered as potential therapies for DMD, this work provides evidence for the variable activation of these systems in different skeletal muscles and the heart. Supported by NIH R01HL104129.
Davi Augusto Mazala, Samuel Andrew English, Dapeng Chen and Eva R Chin
Kinesiology, University of Maryland, College Park, MD
Duchenne muscular dystrophy (DMD) is a progressive degenerative muscle disease that affects 1 out of 3500 boys. It is known that the mouse model most frequently used in DMD research, the mdx mice, upregulates utrophin (utr), a protein with similar roles as dystrophin. Thus, the mdx/utr–/– double mutant (DM) is an alternative model that may be a more appropriate model for studying DMD disease mechanisms. One proposed mechanism behind the disease progression is related to poor intracellular calcium ([Ca2+]i) handling. The purpose of the current study was to evaluate the [Ca2+]i levels in response to repeated tetanic stimulation in intact single muscle fibres from the DM mouse model in comparison to fibres from mdx mice. Single muscle fibres were obtained from the flexor digitorum brevis by collagenase digestion and loaded with Fura-2 AM for [Ca2+]i assessment at a range of stimulation frequencies (10, 30, 50, 70, 100, 120 and 150Hz). There were no differences for peak Fura-2 ratio at any of the stimulation frequencies but the resting Fura-2 ratio was reduced in the DM compared to the mdx mice (0.361±0.029 vs 0.381±0.019; p<0.05). The diameter and length of muscle fibres from DM were smaller compared to the mdx group (p<0.05). Furthermore, 65% of the fibres from DM animals failed to maintain peak Ca2+ during a tetanus, while 43% of the mdx fibres failed. These data suggest that alterations in the Ca2+ handling properties may be due to plasma or t-tubule membrane conductance abnormalities and/or impaired Ca2+ release or storage by the sarcoplasmic reticulum. This project has been funded by Department of Kinesiology Graduate Research Initiative Project (GRIP).
Jessica Delores Gumerson1, Zhyldyz T Kabaeva1, Carol S Davis1, Jeswin B John1, John A Faulkner1,2 and Daniel E Michele1,3
1 Department of Molecular &
Integrative Physiology
2 Department of Biomedical Engineering
3 Department of Internal Medicine, University of Michigan, Ann Arbor,
MI
Susceptibility of muscle to contraction-induced (CI) injury is considered a
hallmark of dystrophin glycoprotein complex associated muscular dystrophies. We
investigated how loss of dystroglycan matrix receptor function in
glycosylation-deficient mice impacted muscle contractile function. Soleus and
extensor digitorum longus (EDL) muscles from Largemyd/myd mice both
demonstrated reduced specific force but surprisingly, only the EDL muscle was
more susceptible to lengthening CI injury. Largemyd/myd soleus muscle
showed no increase in force deficit when compared to WT soleus muscle but still
showed histological features of dystrophic pathology similar to fast muscle.
This suggests that increased CI injury is not the primary mechanism for the
observed muscle degeneration. One possible explanation for the lack of increased
CI injury is that soleus muscle exhibits higher expression of an alternative
matrix receptor,
7β1
integrin. Interestingly, Largemyd/myd dystrophic fast muscles show
fiber type conversion indicated by increased slow-twitch and hybrid fibers, and
a concomitant increase in β1 integrin expression compared to WT muscle.
Therefore, slow fiber type conversion in dystrophic muscle and a corresponding
upregulation of β1 integrin expression might be an important compensatory
mechanism in dystrophic muscle that could be targeted for prevention of muscle
injury.
Justin Percival1, Nicholas Whitehead1, Michael Siegel2, Heidi Gray3, Candace Adamo3, David Marcinek2, Joseph Beavo3 and Stanley Froehner1
1 Physiology and Biophysics
2 Radiology
3 Pharmacology, University of Washington, Seattle, WA
Duchenne Muscular Dystrophy (DMD) is a recessive X–linked neuromuscular disease that results from mutations in dystrophin. The majority of premature deaths of DMD patients result from respiratory failure due to progressive fibrosis that diminishes diaphragm function, while cardiac failure accounts for the remainder of deaths. The loss of functional dystrophin also leads to defective nitric oxide (NO) signaling in skeletal, cardiac and smooth muscle systems. We recently showed that systemic amplification of NO signaling with sildenafil (a phosphodiesterase 5 [PDE5] inhibitor) reversed cardiac dysfunction in the mdx mouse model of DMD. We now show that skeletal muscle cell lines and homogenates contain a pool of active PDE5 and that sildenafil significantly enhanced mdx respiratory muscle (diaphragm) health by reducing fibrosis and Evans Blue Dye uptake, while enhancing contractile force output. In contrast, sildenafil neither affected mdx hindlimb pathology or function nor impacted standard biomarkers of dystrophic disease. Nevertheless, sildenafil treatment appeared to enhance mitochondrial oxidative phosphorylation capacity in mdx hindlimb muscles in vivo, consistent with reported protective effects of enhanced NO-cGMP signaling on mitochondria. In summary, sildenafil may enhance skeletal muscle mitochondrial energetics and may be therapeutically useful in reducing fibrosis and enhancing respiratory muscle function in DMD patients.
Erin Simons1, Dan Brereton2 and Jeffrey Plochocki2
1 Anatomy, Midwestern University,
Glendale, AZ
2 Midwestern University, Glendale, AZ
Dystrophin is a cytoplasmic protein that connects muscle cells to the extracellular matrix. When absent, muscle degeneration and fibrosis occur that resembles the histopathological presentation of Duchenne muscular dystrophy. In mice that lack dystrophin, there is muscle degeneration and regeneration in the first few months of life, with fibrosis increasing after that. In humans muscular dystrophy causes scoliosis of the spine, which presents as pathological kyphosis in mice. In this study, we test the hypothesis that voluntary exercise reduces kyphosis and fibrosis of muscle tissue in dystrophin-deficient mice. The kyphosis was evaluated using the kyphotic index, a standard measure of kyphosis. Contrary to prediction, exercised mice exhibited significantly greater thoracokyphosis than non-exercised mice (p < 0.05). The degree of fibrosis was assessed using a picrosirius red stain. Exercised mice exhibited significantly more fibrosis in the spinal muscles than non-exercised mice (p < 0.05). Our findings suggest exercise accelerates the degeneration and fibrosis of the spinal muscles and the development of pathological kyphosis.
Yi-Wen Chen1,2, Vishakha Sharma1,2, Jigna Narola1, Naoe Harafuji1 and Rongye Shi1
1 Research Center for Genetic
Medicine, Children’s National Medical Center, Washington, DC
2 Department of Integrative Systems Biology, George Washington
University, Washington, DC
Facioscapulohumeral muscular dystrophy (FSHD) is linked to the deletion of the D4Z4 arrays at chromosome 4q35 subtelomeric region. Each D4Z4 array contains a double homeobox 4 (DUX4) open reading frame. We previously showed that the DUX4 functioned as a transcription factor and regulated expression of paired-like homeodomain transcription factor 1 (PITX1) which was identified specifically upregulated in FSHD by expression profiling. In this study, we identified additional downstream targets and regulatory pathways of DUX4 by expression profiling C2C12 myoblasts transfected with expression vectors of DUX4. Expression vectors of DUX4 homologues, namely DUX4c and DUX1, and insertless vectors were used as controls. The expression profiling study was conducted using the Affymetrix 430_2 microarrays. The data was analyzed using Affymetrix MAS 5.0 followed by t test using Genespring GX11. We identified 948 genes to be differentially expressed in the cells expressing the DUX4 gene compared to the insertless control (p < 0.05), while 787 and 901 genes were differentially expressed in cells transfected with DUX4c and DUX1 expression vectors, respectively. The 5 top ranked canonical pathways affected by DUX4 identified using the Ingenuity Pathway Analysis were IGF-1 signaling, ovarian cancer signaling, PTEN signaling, estrogen dependent breast cancer signaling and docosahexaenoic acid signaling. The data showed that DUX4 over-expression lead to activation of pathways involved in cell cycle, proliferation, and apoptosis. The down-regulation of genes involved in IGF1 pathway and the activation of the PTEN pathway suggested a negative regulatory effect of DUX4 on myofiber differentiation, which might contribute to the FSHD pathology.
John M Lawler1, Jeff M Hord2, Yang Lee2, Kumar Joshi1 and Jong-Hee Kim2
1 Health & Kinesiology, Biomedical
Engineering
2 Health & Kinesiology, Texas A&M University, College Station, TX
Duchenne muscular dystrophy (DMD) elicits damage and inflammation in
respiratory (e.g., diaphragm) and limb muscles, and is a caused by an x-linked
mutation in the gene encoding for dystrophin, a scaffolding protein of the
dystroglycan complex (DGC). While reactive oxygen species (ROS) amplify damage
and inflammation, mechanistic links remain elusive. Recent evidence indicates
that impaired integrity of the DGC and muscle damage with DMD are dependent upon
upregulation of caveolin-3 and matrix metalloproteinase-9 (MMP-9), with NAD(P)H
oxidase (Nox) as an ROS source. We tested the hypothesis that elevation of
caveolin-3 and MMP-9 and dislocation of DGC proteins (a-syntrophin, nNOS) were
redox-dependent in the diaphragm of mdx mice. We used a salen-manganese
mimetic of SOD and catalase (EUK-134) and dissociation of Nox subunits (p47phox,
gp91phox) via apocynin as interventions. 20 day old wild-type and mdx
mice were assigned to the following groups for 8 days: wild-type, mdx +
saline, mdx + 30 ml/kg/day EUK-134, mdx + apocynin (1.5 mM in
H2O). EUK-134 attenuated oxidative stress as well as MMP-9 and caveolin-3-positive
staining in the mdx diaphragm. EUK-134 also partially relocalized
α-syntrophin and nNOS in the sarcolemma. Apocynin reduced elevated sarcolemmal
localization of p47phox and MMP-9. These data indicate that redox regulation
exacerbates disruption of the DGC with DMD. Supported by NIH (AR054084
Katrin Hollinger, Delphine Gardan-Salmon, Jenna Dixon, Steven Lonergan and Joshua T Selsby
Animal Science, Iowa State University, Ames, IA
Duchenne muscular dystrophy is characterized by profound muscle injury and is caused by the production of an aberrant dystrophin gene product. In dystrophic muscle peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha appears to reduce muscle injury by increasing expression of the dystrophin-related protein, utrophin, as well as expression of oxidative proteins. The goal of the current study was to determine the extent to which PGC-1alpha over-expression altered the dystrophic proteome. Neonatal mdx mice (n=6) were injected in the right hind limb with adeno- associated virus (AAV) driving PGC-1alpha expression and the left hind limb was injected with an empty AAV vector. After 6 weeks the gastrocnemius was removed, total protein extracted, 2D- DIGE performed, and spots differing in abundance analyzed by mass spectroscopy. Forty-five spots in the 12% gel and 22 spots in the 8% gel were differentially expressed and 16 different proteins were identified. Limbs over-expressing PGC-1alpha had lower expression of serotransferrin indicating reduced pro-oxidant free iron. Additionally, expression of cytoskeletal components was decreased in PGC-1alpha over-expressing muscle suggesting that induced utrophin expression stabilized the muscle membrane. These data support the beneficial role of PGC-1alpha and provide additional insight regarding the underlying mechanism of protection.
Marc Tuazon1 and Gregory C. Henderson2
1 Department of Nutritional
Sciences
2 Department of Exercise Science and Sport Studies, Rutgers
University, New Brunswick, NJ
Mdx mice are a model for Duchenne muscular dystrophy (DMD) and exhibit
increased oxidative stress and inflammation. We sought to determine if the
profile of fatty acids (FAs) in muscle phospholipid (PL) is altered in manners
that could impact anti-inflammatory status or other aspects of muscular function.
8 mdx mice and 8 C57BL/10 control mice (con), on the same standard diet, were
tested with a grip strength meter and then skeletal muscle was collected. The FA
profile of quadriceps PL was determined by HPLC, assessing the abundance of 22
FAs as percentage of total FAs in PL. Compared to con, mdx PL contained less of
the
-3
FA docosahexaenoic acid (DHA) (20.3±1.2% vs. 24.0±1.8%, P<0.05) and more
linoleic acid (13.4±0.5% vs. 11.7±0.9%, P<0.05). Mdx PL also contained more
oleic acid (6.1±1.2% vs. 3.4±0.4%, P<0.05) with no significant difference for
stearic acid; thus,
-9
desaturase activity may have been accentuated in mdx mice. In addition,
-linolenic
acid content in PL was positively correlated with muscular strength in mdx mice
(R=0.83, P<0.05) but not in control mice (R=–0.66, P=0.08), indicating a
potential benefit of
-3
FAs to diseased muscle. In conclusion,
-3
FAs in muscle could be of importance for coping with the disease process in DMD.
Furthermore, we discovered potential evidence for altered desaturase activity
that requires further study to indicate a physiological role.
Supported by Rutgers University
Vladimir Ljubicic, John A Lunde, Louise Boudreault, Shiemaa Khogali, Matthew Burt, Jean-Marc Renaud and Bernard J Jasmin
Cellular and Molecular Medicine, and Centre for Neuromuscular Disease, University of Ottawa, Ottawa, ON, Canada
A therapeutic approach for Duchenne muscular dystrophy (DMD) is to upregulate
utrophin levels in skeletal muscle in an effort to compensate for the lack of
dystrophin. We have previously hypothesized that promotion of the slow,
oxidative myogenic program, which triggers utrophin upregulation, can attenuate
the dystrophic pathology in mdx animals, the murine model of DMD. Indeed,
treatment of mdx mice with the PPAR
activator GW501516
proteins were augmented in response to AICAR, concomitant with a reduction in
RIP140. Exercise increased utrophin A, PGC-1,
and PPAR
mRNAs. This effect was attenuated with AICAR. Our data suggest that AICAR-evoked
muscle plasticity results in beneficial phenotypic adaptations in mdx mice.
Supported by MDA (USA), CIHR and NSERC.
Stephanie Marie Rose Duguez, Helen Johnston, Douglas Johnson, Kristy Brown, Yetrib Hathout and Terence Partridge
Center for Genetic Medicine, Children’s National medical Center, Washington, DC
The generally accepted view of the pathology of Duchenne muscular dystrophy is that absence of dystrophin renders myofibres abnormally susceptible to work-induced trauma, precipitating recurrent bouts of segmental myofibre necrosis. Necrosis stimulates inflammation and a regenerative response by myoblasts; the latter gradually becoming ineffective due to exhaustion of cells and accumulation of connective tissue driven by chronic inflammation. However, while searching for biomarkers in tissue cultures of dystrophic mouse muscle, we found phenomena that cause us to revise this picture. Using stable isotope labeled amino acids (SILAC) combined with liquid chromatography tandem mass spectrometry, we found that undamaged dystrophic myotubes are hypersecretive, excessively effluxing 57 intracellular proteins by mechanisms that cannot be identified as cell death, or generalized increase in passive leakiness. In addition dystrophic myotubes were smaller than wild-type and contain less protein and RNA per nucleus. We hypothesize that lack of dystrophin, as well as predisposing the myofibre to necrosis during physiological stress, has more profound general effects on its metabolic balance and its relationship with the extracellular environment, these potentially contributing to progressive muscle pathology. We identify myosin light chain 1 as a biomarker for in vitro drug screening. Supported by DoD.
Nevenka Juretic, Gonzalo Jorquera, Enrique Jaimovich and Nora Riveros
Centro de Estudios Moleculares de la Célula, Universidad de Chile, Santiago, Chile
Duchenne muscular dystrophy is a neuromuscular disease originated by mutations in the dystrophin gene. Neuregulin-1 (NRG-1) is a growth factor that induces utrophin, a dystrophin homolog, expression. NRG-1β is one of 150 differentially expressed genes in dystrophic (RCDMD) human skeletal muscle cells after electrical stimulation (ES; 400 pulses, 1 ms, 45 Hz). ES increases NRG-1β mRNA and protein levels in RCDMD cells, but has no effect on normal cells.
We investigated the involvement of Ca2+ and PKC isoforms on NRG-1β expression in electrically stimulated RCDMD cells. NRG-1β gene expression was inhibited in the presence of the intracellular Ca2+ chelator BAPTA-AM, and by inhibitors of IP3-dependent slow Ca2+ transients as 2 APB, Ly 294002 and xestospongin B. Ryanodine, a fast Ca2+ signal inhibitor, had no effect. Both BIM VI (general inhibitor of PKC isoforms) and Gö 6976 (specific inhibitor of Ca2+ dependent PKC isoforms) abolished NRG-1β mRNA induction.
Our results suggest that slow Ca2+ signals stimulate NRG-1β transcription in RCDMD cells after depolarization, and that a Ca2+ dependent PKC isoform is needed for activation of NRG-1β expression. Since utrophin can partly compensate dystrophin disfunction, knowledge on the mechanism involved on NRG-1 up regulation could help development of new therapies.
PSD24, FONDAP 15010006, FONDECYT 11100267
transfection restores early mitochondrial functional abnormalities in mdx
skeletal muscle Frederic Nicolas Daussin1, Richard Godin1, Alexis Ascah1, Sonia Deschênes1, Basil Petrof2 and Yan Burelle1
1 Kinésiologie, Université de
Montréal, Montréal, QC, Canada
2 McGill University, Montréal, QC, Canada
We examined the mitochondrial phenotype in skeletal muscle in the early phase
of Duchenne muscular dystrophy (DMD), and determined whether upregulation of
mitochondrial biogenesis via PGC-1
transfection is beneficial in the mdx mice, a murine model of DMD. Compared to
wild-type, 6 weeks-old mdx mice exhibited mitochondrial dysfunction including a
lower oxidative capacity, a higher susceptibility to Ca2+-induced PTP opening,
and an adaptive increase in ROS buffering capacity. Electroporation of the PGC-1
plasmid largely restored mitochondrial density, as assessed by several marker
proteins. Importantly, this translated into an increased mitochondrial Ca2+
buffering capacity and enhanced resistance to PTP opening. Overall, this study
reveals several mitochondrial functional abnormalities in the early phase of the
disease, which were ameliorated 7 days after PGC-1
transfection. In particular, amelioration of mitochondrial Ca2+ buffering
capacity may help to improve cellular Ca2+ regulation by limiting the adverse
effect of excessive calcium levels, which characterizes DMD.
Jeffrey H Plochocki1, Jeffrey M Costas2 and Renita Ho2
1 Anatomy, Midwestern University,
Glendale, AZ
2 Midwestern University, Glendale, AZ
Dilated cardiomyopathy is a leading cause of death in patients with muscular dystrophy (MD). Although there is no cure for MD, exercise and corticosteroids (CS) are commonly prescribed to prolong cardiac function. To test the affects of these treatments on cardiac mass and fibrosis, dystrophin deficient (mdx) mice were separated into control and treatment groups. Additionally, mice were treated with an agonist for the serotonin 5-HT2b receptor, which is known to play a role in cardiac hypertrophy. The 5-HT2br agonist was the only treatment that significantly elevated cardiac mass and left ventricular wall thickness above controls, while reducing cardiac fibrosis (P < 0.05). Mice treated with voluntary running exercise exhibited significantly reduced fibrosis compared to controls, but similar cardiac size. CS treatment and CS treatment combined with exercise was not effective at reducing fibrosis and maintaining cardiac mass. Quadriceps fibrosis was significantly diminished only in the CS treated mice relative to controls. Our findings suggest 5-HT2br agonists may maintain cardiac mass in dystrophin deficient mice and should be further investigated as a treatment modality for MD.
Ellen Yukie Fukuda1, Renato Davino Chiovatto1, David Feder1, Maira Lazzarini Guimarães Pereira2, Ivan Rodrigues Barros Godoy2, Cledson Silveira Silva2, Diego Nogueira Silvestre2, Viviana Boccardi Palou2, Alzira Alves de Siqueira Carvalho2, Rosangela Aparecida Santos3, Maria Helena Catelli de Carvalho3, Cristina Zotti Nassis1 and Christine Carrico4
1 Pharmacology, ABC School of
Medicine, Santo André, Brazil
2 ABC School of Medicine, Santo André, Brazil
3 São Paulo University, São Paulo, Brazil
4 ASPET, Bethesda, MD
This study was designed to assess and characterize the steroidal hormone receptors in skeletal muscle of dystrophic mice. Mdx mice are a model of Xp21 dystrophin-deficient muscular dystrophy. Five mdx and five C57BL6 (control) mice four months old were used. Muscles, including heart, gastrocnemius, diaphragm, quadriceps, soleus and extensorum digitorum longus (EDL) were dissected, frozen in liquid nitrogen. PCR amplification was carried out to assess the steroidal receptors mRNA expression. Mdx mice showed significant raise in the expression of steroidal receptors mRNA when compared to the C57BL6 mice: androgens receptors in heart, alpha estrogen receptors in EDL and beta estrogen receptors in quadriceps were increased. In addition, significant lowered levels of some other hormone receptors were found: corticosteroid receptors in EDL and alpha estrogen receptors in quadriceps, leading to the conclusion that dystrophic muscles disclose significant differences on hormone receptors, when compared to C57BL6 strain. There was no support for this study.
Mathieu St-Louis1,2, Solecki Michal1 and Robin N Michel1,2
1 Exercise Science
2 Chemistry and Biochemistry, Concordia University, Montreal, QC,
Canada
Myostatin (Mstn) is a negative muscle mass regulator part of the TGF-ß family of growth factors. The increase in muscle mass is a result of hypertrophy and hyperplasia. To investigate the role for Mstn in muscle fiber remodeling, we compared the effects of functional muscle overload (OV) of the plantaris in Mstn Knockout (Mstn –/–) and C57BL/6 (WT) mice. The muscle midbelly area of non-OV Mstn–/– mice was double that of WT counterparts due to a higher number of muscle fibers and fibers displaying greater cross sectional size. Six weeks of functional overload led to a blunted growth marked by significant differences in the increase in mean midbelly area (WT: 90%; Mstn–/–: 23%; p<0.05). The blunted growth was concomitant with increases in individual fiber sizes (WT: 35%; Mstn–/–:8%; p<0.05). Additionally, muscles from Mstn–/– mice maintain the plasticity to adapt to functional demands as marked by changes in the expression of the different isoforms of MHC, however, that response was also blunted. We found that he distal portion of the plantaris muscle was a region of major remodeling in both groups. Mstn–/– mice displayed more damage at all levels analyzed after one week where WT and Mstn–/– mice had in that region high expression of regeneration markers such as MyoD and Myogenin. That remodeling region was bigger in Mstn –/– compared to WT animals. At 2 and 6 weeks of overload, Mstn –/– showed more centrally nucleated fibers compared to WT. All of that is reflecting increased hyperplasia. Functional overload led to a blunted growth and switch to a slower phenotype. This differential growth pattern may be of importance when considering Myostatin-related therapies in aging and diseases such as muscular dystrophy. Funded by CIHR, NSERC and CRC.