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In JoVE (1)
Other Publications (10)
- Archives of Physical Medicine and Rehabilitation
- Journal of Aerosol Medicine and Pulmonary Drug Delivery
- Journal of Neuroscience Methods
- Molecular Biology of the Cell
- The American Journal of Pathology
- American Journal of Physiology. Cell Physiology
- American Journal of Physiology. Cell Physiology
- The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
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Articles by Joseph A. Roche in JoVE
In vivo מכרסמים דגם של פגיעה הנגרמת על התכווצות ולא פולשנית לניטור של התאוששות
Richard M. Lovering1,2, Joseph A. Roche1, Mariah H. Goodall2, Brett B. Clark2, Alan McMillan3
1Department of Physiology, University of Maryland School of Medicine, 2Department of Orthopaedics, University of Maryland School of Medicine, 3Department of Diagnostic Radiology, University of Maryland School of Medicine
Other articles by Joseph A. Roche on PubMed
Archives of Physical Medicine and Rehabilitation. May, 2007 | Pubmed ID: 17466731
To determine if the proliferation of myogenic cells is equally important to recovery of contractile function after 2 different types of contraction-induced muscle injuries.
Identification of Skeletal Muscle Mutations in Tail Snips from Neonatal Mice Using Immunohistochemistry
BioTechniques. Jun, 2007 | Pubmed ID: 17612291
Neuroreport. Oct, 2008 | Pubmed ID: 18815587
The protein, dysferlin, mediates sarcolemmal repair in vitro, implicating defective membrane repair in dysferlinopathies. To study the role of dysferlin in vivo, we assessed contractile function, sarcolemmal integrity, and myogenesis before and after injury from large-strain lengthening contractions in dysferlin-null and control mice. We report that dysferlin-null muscles produce higher contractile torque, and are equally susceptible to initial injury but recover from injury more slowly. Two weeks after injury, control muscles retain fluorescein dextran and do not show myogenesis. Dysferlin-null muscles do not retain fluorescein dextran, and show necrosis followed by myogenesis. Our data indicate that recovery of control muscles from injury primarily involves sarcolemmal repair whereas recovery of dysferlin-null muscles primarily involves myogenesis without repair and long-term survival of myofibers.
Journal of Aerosol Medicine and Pulmonary Drug Delivery. Dec, 2008 | Pubmed ID: 18823258
Amount of drug actually reaching the target region in the lung following pulmonary inhalation is often estimated at less than 10% for older devices. Current particle and device engineering technologies have improved on this but still fail to recover the "wasted" fraction of the drug and deliver it deeper into the lungs, which is generally desirable. FDA has approved several exogenous surfactants for prophylaxis and rescue treatment of respiratory distress syndrome (RDS). Their approved mode of administration (intratracheal instillation) and site of action (alveolar spaces) suggest that the phospholipids in the exogenous surfactants can spread from the trachea to alveolar air spaces and exert advantageous effects. We investigated whether in vivo lung migration of particles based on this phenomenon was possible and could be quantified based on changes in total and regional deposition of fluorescently labeled latex beads, utilized as an insoluble drug model. Following intranasal administration of beads, migration to rodent lungs was monitored upon intranasal instillation of Survanta (exogenous surfactant) or saline (control). After intranasal instillation approximately 12% of beads were found to migrate to the lung, and total lung deposition increased by approximately 10% on administration of Survanta or saline (control). After intranasal administration approximately 1% of beads in the lung were found to migrate to peripheral regions of the lungs, and a four- to six-fold increase in peripheral lung deposition was observed after Survanta instillation, compared to the saline control, which was determined to be independent of dose and volume of Survanta instillate in the range we studied. The in vivo rodent studies provided support for the idea that intranasally administered particles deposited in non-target lung locations may be translocated to peripheral sites in the lung therapeutically after surfactant application.
Journal of Neuroscience Methods. Jul, 2009 | Pubmed ID: 19433107
We used a gait analysis system (GAS) to measure the changes in locomotion parameters of adult Sprague-Dawley rats after neuromuscular injury, induced by repeated large-strain lengthening contractions of the dorsiflexors muscles. We developed a logistic regression model from test runs of control and permanently impaired (denervation of the dorsiflexor muscles) rats and used this model to predict the probabilities of locomotory impairment in rats injured by lengthening contractions. The data showed that GAS predicts the probability of locomotory impairment with very high reliability, with values close to 100% immediately after injury and close to 0% after several weeks of recovery from injury. The six transformed locomotion parameters most effective in the model were in three domains: frequency, force, and time. We conclude that application of the GAS instrument with our predictive model accurately identifies locomotory changes due to neuromuscular deficits. Use of this technology should be valuable for monitoring the progression of a neuromuscular disease and the effects of therapeutic interventions.
The Rho-guanine Nucleotide Exchange Factor Domain of Obscurin Activates RhoA Signaling in Skeletal Muscle
Molecular Biology of the Cell. Sep, 2009 | Pubmed ID: 19605563
Obscurin is a large ( approximately 800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of obscurin's rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of obscurin binds selectively to rhoA, and that rhoA colocalizes with obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of obscurin's rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of obscurin's rhoGEF domain. Our results suggest that obscurin's rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo.
Genetic Manipulation of Dysferlin Expression in Skeletal Muscle: Novel Insights into Muscular Dystrophy
The American Journal of Pathology. Nov, 2009 | Pubmed ID: 19834057
Mutations in the gene DYSF, which codes for the protein dysferlin, underlie Miyoshi myopathy and limb-girdle muscular dystrophy 2B in humans and produce a slowly progressing skeletal muscle degenerative disease in mice. Dysferlin is a Ca(2+)-sensing, regulatory protein that is involved in membrane repair after injury. To assess the function of dysferlin in healthy and dystrophic skeletal muscle, we generated skeletal muscle-specific transgenic mice with threefold overexpression of this protein. These mice were phenotypically indistinguishable from wild-type, and more importantly, the transgene completely rescued the muscular dystrophy (MD) disease in Dysf-null A/J mice. The dysferlin transgene rescued all histopathology and macrophage infiltration in skeletal muscle of Dysf(-/-) A/J mice, as well as promoted the rapid recovery of muscle function after forced lengthening contractions. These results indicate that MD in A/J mice is autonomous to skeletal muscle and not initiated by any other cell type. However, overexpression of dysferlin did not improve dystrophic symptoms or membrane instability in the dystrophin-glycoprotein complex-lacking Scgd (delta-sarcoglycan) null mouse, indicating that dysferlin functionality is not a limiting factor underlying membrane repair in other models of MD. In summary, the restoration of dysferlin in skeletal muscle fibers is sufficient to rescue the MD in Dysf-deficient mice, although its mild overexpression does not appear to functionally enhance membrane repair in other models of MD.
Extensive Mononuclear Infiltration and Myogenesis Characterize Recovery of Dysferlin-null Skeletal Muscle from Contraction-induced Injuries
American Journal of Physiology. Cell Physiology. Feb, 2010 | Pubmed ID: 19923419
We studied the response of dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice.
Physiological and Histological Changes in Skeletal Muscle Following in Vivo Gene Transfer by Electroporation
American Journal of Physiology. Cell Physiology. Nov, 2011 | Pubmed ID: 21832248
Electroporation (EP) is used to transfect skeletal muscle fibers in vivo, but its effects on the structure and function of skeletal muscle tissue have not yet been documented in detail. We studied the changes in contractile function and histology after EP and the influence of the individual steps involved to determine the mechanism of recovery, the extent of myofiber damage, and the efficiency of expression of a green fluorescent protein (GFP) transgene in the tibialis anterior (TA) muscle of adult male C57Bl/6J mice. Immediately after EP, contractile torque decreased by ∼80% from pre-EP levels. Within 3 h, torque recovered to ∼50% but stayed low until day 3. Functional recovery progressed slowly and was complete at day 28. In muscles that were depleted of satellite cells by X-irradiation, torque remained low after day 3, suggesting that myogenesis is necessary for complete recovery. In unirradiated muscle, myogenic activity after EP was confirmed by an increase in fibers with central nuclei or developmental myosin. Damage after EP was confirmed by the presence of necrotic myofibers infiltrated by CD68+ macrophages, which persisted in electroporated muscle for 42 days. Expression of GFP was detected at day 3 after EP and peaked on day 7, with ∼25% of fibers transfected. The number of fibers expressing green fluorescent protein (GFP), the distribution of GFP+ fibers, and the intensity of fluorescence in GFP+ fibers were highly variable. After intramuscular injection alone, or application of the electroporating current without injection, torque decreased by ∼20% and ∼70%, respectively, but secondary damage at D3 and later was minimal. We conclude that EP of murine TA muscles produces variable and modest levels of transgene expression, causes myofiber damage due to the interaction of intramuscular injection with the permeabilizing current, and that full recovery requires myogenesis.
The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society. Nov, 2011 | Pubmed ID: 22043020
Mutations in the DYSF gene that severely reduce the levels of the protein dysferlin are implicated in muscle-wasting syndromes known as dysferlinopathies. Although studies of its function in skeletal muscle have focused on its potential role in repairing the plasma membrane, dysferlin has also been found, albeit inconsistently, in the sarcoplasm of muscle fibers. The aim of this article is to study the localization of dysferlin in skeletal muscle through optimized immunolabeling methods. We studied the localization of dysferlin in control rat skeletal muscle using several different methods of tissue collection and subsequent immunolabeling. We then applied our optimized immunolabeling methods on human cadaveric muscle, control and dystrophic human muscle biopsies, and control and dysferlin-deficient mouse muscle. Our data suggest that dysferlin is present in a reticulum of the sarcoplasm, similar but not identical to those containing the dihydropyridine receptors and distinct from the distribution of the sarcolemmal protein dystrophin. Our data illustrate the importance of tissue fixation and antigen unmasking for proper immunolocalization of dysferlin. They suggest that dysferlin has an important function in the internal membrane systems of skeletal muscle, involved in calcium homeostasis and excitation-contraction coupling.