Three-dimensional imaging studies in mice identify cellular dynamics of skeletal muscle regeneration.

The function of many organs, including skeletal muscle, depends on their three-dimensional structure. Muscle regeneration therefore requires not only reestablishment of myofibers but also restoration of tissue architecture. Resident muscle stem cells (SCs) are essential for regeneration, but how SCs regenerate muscle architecture is largely unknown. We address this problem using genetic labeling of mouse SCs and whole-mount imaging to reconstruct, in three dimensions, muscle regeneration. Unexpectedly, we found that myofibers form via two distinct phases of fusion and the residual basement membrane of necrotic myofibers is critical for promoting fusion and orienting regenerated myofibers. Furthermore, the centralized myonuclei characteristic of regenerated myofibers are associated with myofibrillogenesis and endure months post injury. Finally, we elucidate two cellular mechanisms for the formation of branched myofibers, a pathology characteristic of diseased muscle. We provide a synthesis of the cellular events of regeneration and show that these differ from those used during development.

Myofiber-type-dependent 'boulder' or 'multitudinous pebble' formations across distinct amylopectinoses.

At least five enzymes including three E3 ubiquitin ligases are dedicated to glycogen's spherical structure. Absence of any reverts glycogen to a structure resembling amylopectin of the plant kingdom. This amylopectinosis (polyglucosan body formation) causes fatal neurological diseases including adult polyglucosan body disease (APBD) due to glycogen branching enzyme deficiency, Lafora disease (LD) due to deficiencies of the laforin glycogen phosphatase or the malin E3 ubiquitin ligase and type 1 polyglucosan body myopathy (PGBM1) due to RBCK1 E3 ubiquitin ligase deficiency. Little is known about these enzymes' functions in glycogen structuring. Toward understanding these functions, we undertake a comparative murine study of the amylopectinoses of APBD, LD and PGBM1. We discover that in skeletal muscle, polyglucosan bodies form as two main types, small and multitudinous ('pebbles') or giant and single ('boulders'), and that this is primarily determined by the myofiber types in which they form, 'pebbles' in glycolytic and 'boulders' in oxidative fibers. This pattern recapitulates what is known in the brain in LD, innumerable dust-like in astrocytes and single giant sized in neurons. We also show that oxidative myofibers are relatively protected against amylopectinosis, in part through highly increased glycogen branching enzyme expression. We present evidence of polyglucosan body size-dependent cell necrosis. We show that sex influences amylopectinosis in genotype, brain region and myofiber-type-specific fashion. RBCK1 is a component of the linear ubiquitin chain assembly complex (LUBAC), the only known cellular machinery for head-to-tail linear ubiquitination critical to numerous cellular pathways. We show that the amylopectinosis of RBCK1 deficiency is not due to loss of linear ubiquitination, and that another function of RBCK1 or LUBAC must exist and operate in the shaping of glycogen. This work opens multiple new avenues toward understanding the structural determinants of the mammalian carbohydrate reservoir critical to neurologic and neuromuscular function and disease.

CSTB gene replacement improves neuroinflammation, neurodegeneration and ataxia in murine type 1 progressive myoclonus epilepsy.

EPM1 is the most common form of Progressive Myoclonus Epilepsy characterized by late-childhood onset, ever-worsening and disabling myoclonus, seizures, ataxia, psychiatric disease, and shortened lifespan. EPM1 is caused by expansions of a dodecamer repeat sequence in the promoter of CSTB (cystatin B), which dramatically reduces, but does not eliminate, gene expression. The relatively late onset and consistent presence of a minimal amount of protein product makes EPM1 a favorable target for gene replacement therapy. If treated early, these children's normally developed brains could be rescued from the neurodegeneration that otherwise follows, and their cross-reactive immunological material (CRIM) positive status greatly reduces transgene related toxicity. We performed a proof-of-concept CSTB gene replacement study in Cstb knockout mice by introducing full-length human CSTB driven by the CBh promoter packaged in AAV9 and administered at postnatal days 21 and 60. Mice were sacrificed at 2 or 9 months of age, respectively. We observed significant improvements in expression levels of neuroinflammatory pathway genes and cerebellar granule cell layer apoptosis, as well as amelioration of motor impairment. The data suggest that gene replacement is a promising therapeutic modality for EPM1 and could spare affected children and families the ravages of this otherwise severe neurodegenerative disease.

Tissue Clearing and Confocal Microscopic Imaging for Skeletal Muscle.

Skeletal muscle is a highly ordered tissue composed of a complex network of a diverse variety of cells. The dynamic spatial and temporal interaction between these cells during homeostasis and during times of injury gives the skeletal muscle its regenerative capacity. To properly understand the process of regeneration, a three-dimensional (3-D) imaging process must be conducted. With the advancement of imaging and computing technology, it has become powerful to analyze spatial data from confocal microscope images. In order to prepare whole tissue skeletal muscle samples for confocal imaging, the muscle must be subjected to tissue clearing. With the use of an ideal optical clearing protocol - one that minimizes light scattering via refractive index mismatching - a more accurate 3-D image of the muscle can be produced as it does not involve the physical sectioning of the muscle. While there have been several protocols relating to the study of 3-D biology in whole tissue, these protocols have primarily been focused on the nervous system. In this chapter, we present a new method for skeletal muscle tissue clearing. In addition, this protocol aims to outline the specific parameters required for taking 3-D images of immunofluorescence-stained skeletal muscle samples using a confocal microscope.

Three-Dimensional Imaging Analysis for Skeletal Muscle.

Skeletal muscle is a highly ordered tissue composed of a complex network of a diverse variety of cells. The dynamic spatial and temporal interaction between these cells during homeostasis and during times of injury gives the skeletal muscle its regenerative capacity. In order to properly understand the process of regeneration, a three-dimensional (3-D) imaging process must be conducted. While there have been several protocols studying 3-D imaging, it has primarily been focused on the nervous system. This protocol aims to outline the workflow for rendering a 3-D image of the skeletal muscle using spatial data from confocal microscope images. This protocol uses the ImageJ, Ilastik, and Imaris software for 3-D rendering and computational image analysis as both are relatively easy to use and have powerful segmentation capabilities.

Predicting implementation of active learning by tenure-track teaching faculty using robust cluster analysis.

Background: The University of California system has a novel tenure-track education-focused faculty position called Lecturer with Security of Employment (working titles: Teaching Professor or Professor of Teaching). We focus on the potential difference in implementation of active-learning strategies by faculty type, including tenure-track education-focused faculty, tenure-track research-focused faculty, and non-tenure-track lecturers. In addition, we consider other instructor characteristics (faculty rank, years of teaching, and gender) and classroom characteristics (campus, discipline, and class size). We use a robust clustering algorithm to determine the number of clusters, identify instructors using active learning, and to understand the instructor and classroom characteristics in relation to the adoption of active-learning strategies. Results: We observed 125 science, technology, engineering, and mathematics (STEM) undergraduate courses at three University of California campuses using the Classroom Observation Protocol for Undergraduate STEM to examine active-learning strategies implemented in the classroom. Tenure-track education-focused faculty are more likely to teach with active-learning strategies compared to tenure-track research-focused faculty. Instructor and classroom characteristics that are also related to active learning include campus, discipline, and class size. The campus with initiatives and programs to support undergraduate STEM education is more likely to have instructors who adopt active-learning strategies. There is no difference in instructors in the Biological Sciences, Engineering, or Information and Computer Sciences disciplines who teach actively. However, instructors in the Physical Sciences are less likely to teach actively. Smaller class sizes also tend to have instructors who teach more actively. Conclusions: The novel tenure-track education-focused faculty position within the University of California system represents a formal structure that results in higher adoption of active-learning strategies in undergraduate STEM education. Campus context and evolving expectations of the position (faculty rank) contribute to the symbols related to learning and teaching that correlate with differential implementation of active learning. Supplementary Information: The online version contains supplementary material available at 10.1186/s40594-022-00365-9.

High frequency of an otherwise rare phenotype in a small and isolated tiger population.

Most endangered species exist today in small populations, many of which are isolated. Evolution in such populations is largely governed by genetic drift. Empirical evidence for drift affecting striking phenotypes based on substantial genetic data are rare. Approximately 37% of tigers (Panthera tigris) in the Similipal Tiger Reserve (in eastern India) are pseudomelanistic, characterized by wide, merged stripes. Camera trap data across the tiger range revealed the presence of pseudomelanistic tigers only in Similipal. We investigated the genetic basis for pseudomelanism and examined the role of drift in driving this phenotype's frequency. Whole-genome data and pedigree-based association analyses from captive tigers revealed that pseudomelanism cosegregates with a conserved and functionally important coding alteration in Transmembrane Aminopeptidase Q (Taqpep), a gene responsible for similar traits in other felid species. Noninvasive sampling of tigers revealed a high frequency of the Taqpep p.H454Y mutation in Similipal (12 individuals, allele frequency = 0.58) and absence from all other tiger populations (395 individuals). Population genetic analyses confirmed few (minimal number) tigers in Similipal, and its genetic isolation, with poor geneflow. Pairwise FST (0.33) at the mutation site was high but not an outlier. Similipal tigers had low diversity at 81 single nucleotide polymorphisms (mean heterozygosity = 0.28, SD = 0.27). Simulations were consistent with founding events and drift as possible drivers for the observed stark difference of allele frequency. Our results highlight the role of stochastic processes in the evolution of rare phenotypes. We highlight an unusual evolutionary trajectory in a small and isolated population of an endangered species.

VEGFR-1/Flt-1 inhibition increases angiogenesis and improves muscle function in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is characterized by structural degeneration of muscle, which is exacerbated by localized functional ischemia due to loss of nitric oxide synthase-induced vasodilation. Treatment strategies aimed at increasing vascular perfusion have been proposed. Toward this end, we have developed monoclonal antibodies (mAbs) that bind to the vascular endothelial growth factor (VEGF) receptor VEGFR-1 (Flt-1) and its soluble splice variant isoform (sFlt-1) leading to increased levels of free VEGF and proangiogenic signaling. The lead chimeric mAb, 21B3, had high affinity and specificity for both human and mouse sFlt-1 and inhibited VEGF binding to sFlt-1 in a competitive manner. Proof-of-concept studies in the mdx mouse model of Duchenne muscular dystrophy showed that intravenous administration of 21B3 led to elevated VEGF levels, increased vascularization and blood flow to muscles, and decreased fibrosis after 6-12 weeks of treatment. Greater muscle strength was also observed after 4 weeks of treatment. A humanized form of the mAb, 27H6, was engineered and demonstrated a comparable pharmacologic effect. Overall, administration of anti-Flt-1 mAbs in mdx mice inhibited the VEGF:Flt-1 interaction, promoted angiogenesis, and improved muscle function. These studies suggest a potential therapeutic benefit of Flt-1 inhibition for patients with Duchenne muscular dystrophy.

Comparison of Cluster Analysis Methodologies for Characterization of Classroom Observation Protocol for Undergraduate STEM (COPUS) Data.

The Classroom Observation Protocol for Undergraduate STEM (COPUS) provides descriptive feedback to instructors by capturing student and instructor behaviors occurring in the classroom. Due to the increasing prevalence of COPUS data collection, it is important to recognize how researchers determine whether groups of courses or instructors have unique classroom characteristics. One approach uses cluster analysis, highlighted by a recently developed tool, the COPUS Analyzer, that enables the characterization of COPUS data into one of seven clusters representing three groups of instructional styles (didactic, interactive, and student centered). Here, we examine a novel 250 course data set and present evidence that a predictive cluster analysis tool may not be appropriate for analyzing COPUS data. We perform a de novo cluster analysis and compare results with the COPUS Analyzer output and identify several contrasting outcomes regarding course characterizations. Additionally, we present two ensemble clustering algorithms: 1) k-means and 2) partitioning around medoids. Both ensemble algorithms categorize our classroom observation data into one of two clusters: traditional lecture or active learning. Finally, we discuss implications of these findings for education research studies that leverage COPUS data.

Transcriptional and cytopathological hallmarks of FSHD in chronic DUX4-expressing mice.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by loss of repression of the DUX4 gene; however, the DUX4 protein is rare and difficult to detect in human muscle biopsies, and pathological mechanisms are obscure. FSHD is also a chronic disease that progresses slowly over decades. We used the sporadic, low-level, muscle-specific expression of DUX4 enabled by the iDUX4pA-HSA mouse to develop a chronic long-term muscle disease model. After 6 months of extremely low sporadic DUX4 expression, dystrophic muscle presented hallmarks of FSHD histopathology, including muscle degeneration, capillary loss, fibrosis, and atrophy. We investigated the transcriptional profile of whole muscle as well as endothelial cells and fibroadiopogenic progenitors (FAPs). Strikingly, differential gene expression profiles of both whole muscle and, to a lesser extent, FAPs, showed significant overlap with transcriptional profiles of MRI-guided human FSHD muscle biopsies. These results demonstrate a pathophysiological similarity between disease in muscles of iDUX4pA-HSA mice and humans with FSHD, solidifying the value of chronic rare DUX4 expression in mice for modeling pathological mechanisms in FSHD and highlighting the importance FAPs in this disease.

Inhibition of FLT1 ameliorates muscular dystrophy phenotype by increased vasculature in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease in which the dystrophin coding for a membrane stabilizing protein is mutated. Recently, the vasculature has also shown to be perturbed in DMD and DMD model mdx mice. Recent DMD transcriptomics revealed the defects were correlated to a vascular endothelial growth factor (VEGF) signaling pathway. To reveal the relationship between DMD and VEGF signaling, mdx mice were crossed with constitutive (CAGCreERTM:Flt1LoxP/LoxP) and endothelial cell-specific conditional gene knockout mice (Cdh5CreERT2:Flt1LoxP/LoxP) for Flt1 (VEGFR1) which is a decoy receptor for VEGF. Here, we showed that while constitutive deletion of Flt1 is detrimental to the skeletal muscle function, endothelial cell-specific Flt1 deletion resulted in increased vascular density, increased satellite cell number and improvement in the DMD-associated phenotype in the mdx mice. These decreases in pathology, including improved muscle histology and function, were recapitulated in mdx mice given anti-FLT1 peptides or monoclonal antibodies, which blocked VEGF-FLT1 binding. The histological and functional improvement of dystrophic muscle by FLT1 blockade provides a novel pharmacological strategy for the potential treatment of DMD.

Inhibition of microRNA-92a increases blood vessels and satellite cells in skeletal muscle but does not improve duchenne muscular dystrophy-related phenotype in mdx mice.

INTRODUCTION: The vasculature and blood flow in muscle are perturbed in Duchenne muscular dystrophy (DMD) and its mdx mouse model. MicroRNA-92a (miR-92a) is enriched in endothelial cells, especially during ischemic injury. METHODS: Because antagonizing miR-92a was shown to result in increased proliferation and migration of endothelial cells and recovery from ischemia, we assessed the effects of Antagomir-92a in vitro in muscle stem cell culture and in vivo in mdx mice. RESULTS: miR-92a was found to be highly expressed in muscle endothelial cells and satellite cells. Treatment with Antagomir-92a increased capillary density and tissue perfusion, which was accompanied by an increase in satellite cells. However, Antagomir-92a-treated mdx mice showed no histological improvement and had worse muscle function. Antagomir-92a suppressed myogenic differentiation in satellite cell culture. DISCUSSION: AntagomiR-92a improves the vasculature but not the muscle in mdx mice, possibly due to its side effects on satellite cell differentiation. Muscle Nerve 59:594-594, 2019.

Isometric resistance training increases strength and alters histopathology of dystrophin-deficient mouse skeletal muscle.

Mutation to the dystrophin gene causes skeletal muscle weakness in patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). Deliberation continues regarding implications of prescribing exercise for these patients. The purpose of this study was to determine whether isometric resistance exercise (~10 tetanic contractions/session) improves skeletal muscle strength and histopathology in the mdx mouse model of DMD. Three isometric training sessions increased in vivo isometric torque (22%) and contractility rates (54%) of anterior crural muscles of mdx mice. Mice expressing a BMD-causing missense mutated dystrophin on the mdx background showed comparable increases in torque (22%), while wild-type mice showed less change (11%). Increases in muscle function occurred within 1 h and peaked 3 days posttraining; however, the adaptation was lost after 7 days unless retrained. Six isometric training sessions over 4 wk caused increased isometric torque (28%) and contractility rates (22-28%), reduced fibrosis, as well as greater uniformity of fiber cross-sectional areas, fewer embryonic myosin heavy-chain-positive fibers, and more satellite cells in tibialis anterior muscle compared with the contralateral untrained muscle. Ex vivo functional analysis of isolated extensor digitorum longus (EDL) muscle from the trained hindlimb revealed greater absolute isometric force, lower passive stiffness, and a lower susceptibility to eccentric contraction-induced force loss compared with untrained EDL muscle. Overall, these data support the concept that exercise training in the form of isometric tetanic contractions can improve contractile function of dystrophin-deficient muscle, indicating a potential role for enhancing muscle strength in patients with DMD and BMD. NEW & NOTEWORTHY We focused on adaptive responses of dystrophin-deficient mouse skeletal muscle to isometric contraction training and report that in the absence of dystrophin (or in the presence of a mutated dystrophin), strength and muscle histopathology are improved. Results suggest that the strength gains are associated with fiber hypertrophy, reduced fibrosis, increased number of satellite cells, and blunted eccentric contraction-induced force loss in vitro. Importantly, there was no indication that the isometric exercise training was deleterious to dystrophin-deficient muscle.

Muscle Satellite Cell Cross-Talk with a Vascular Niche Maintains Quiescence via VEGF and Notch Signaling.

Skeletal muscle is a complex tissue containing tissue resident muscle stem cells (satellite cells) (MuSCs) important for postnatal muscle growth and regeneration. Quantitative analysis of the biological function of MuSCs and the molecular pathways responsible for a potential juxtavascular niche for MuSCs is currently lacking. We utilized fluorescent reporter mice and muscle tissue clearing to investigate the proximity of MuSCs to capillaries in 3 dimensions. We show that MuSCs express abundant VEGFA, which recruits endothelial cells (ECs) in vitro, whereas blocking VEGFA using both a vascular endothelial growth factor (VEGF) inhibitor and MuSC-specific VEGFA gene deletion reduces the proximity of MuSCs to capillaries. Importantly, this proximity to the blood vessels was associated with MuSC self-renewal in which the EC-derived Notch ligand Dll4 induces quiescence in MuSCs. We hypothesize that MuSCs recruit capillary ECs via VEGFA, and in return, ECs maintain MuSC quiescence though Dll4.

CD301b/MGL2+ Mononuclear Phagocytes Orchestrate Autoimmune Cardiac Valve Inflammation and Fibrosis.

BACKGROUND: Valvular heart disease is common and affects the mitral valve (MV) most frequently. Despite the prevalence of MV disease (MVD), the cellular and molecular pathways that initiate and perpetuate it are not well understood. METHODS: K/B.g7 T-cell receptor transgenic mice spontaneously develop systemic autoantibody-associated autoimmunity, leading to fully penetrant fibroinflammatory MVD and arthritis. We used multiparameter flow cytometry, intracellular cytokine staining, and immunofluorescent staining to characterize the cells in inflamed K/B.g7 MVs. We used genetic approaches to study the contribution of mononuclear phagocytes (MNPs) to MVD in this model. Specifically, we generated K/B.g7 mice in which either CX3CR1 or CD301b/macrophage galactose N-acetylgalactosamine-specific lectin 2 (MGL2)-expressing MNPs were ablated. Using K/B.g7 mice expressing Cx3Cr1-Cre, we conditionally deleted critical inflammatory molecules from MNPs, including the Fc-receptor signal-transducing tyrosine kinase Syk and the cell adhesion molecule very late antigen-4. We performed complementary studies using monoclonal antibodies to block key inflammatory molecules. We generated bone marrow chimeric mice to define the origin of the inflammatory cells present in the MV and to determine which valve cells respond to the proinflammatory cytokine tumor necrosis factor (TNF). Finally, we examined specimens from patients with rheumatic heart disease to correlate our findings to human pathology. RESULTS: MNPs comprised the vast majority of MV-infiltrating cells; these MNPs expressed CX3CR1 and CD301b/MGL2. Analogous cells were present in human rheumatic heart disease valves. K/B.g7 mice lacking CX3CR1 or in which CD301b/MGL2-expressing MNPs were ablated were protected from MVD. The valve-infiltrating CD301b/MGL2+ MNPs expressed tissue-reparative molecules including arginase-1 and resistin-like molecule α. These MNPs also expressed the proinflammatory cytokines TNF and interleukin-6, and antibody blockade of these cytokines prevented MVD. Deleting Syk from CX3CR1-expressing MNPs reduced their TNF and interleukin-6 production and also prevented MVD. TNF acted through TNF receptor-1 expressed on valve-resident cells to increase the expression of vascular cell adhesion molecule-1. Conditionally deleting the vascular cell adhesion molecule-1 ligand very late antigen-4 from CX3CR1-expressing MNPs prevented MVD. CONCLUSIONS: CD301b/MGL2+ MNPs are key drivers of autoimmune MVD in K/B.g7 mice and are also present in human rheumatic heart disease. We define key inflammatory molecules that drive MVD in this model, including Syk, TNF, interleukin-6, very late antigen-4, and vascular cell adhesion molecule-1.

Extraocular Muscle Repair and Regeneration.

PURPOSE OF REVIEW: The goal of this review is to summarize the unique regenerative milieu within mature mammalian extraocular muscles (EOMs). This will aid in understanding disease propensity for and sparing of EOMs in skeletal muscle diseases as well as the recalcitrance of the EOM to injury. RECENT FINDINGS: The EOMs continually remodel throughout life and contain an extremely enriched number of myogenic precursor cells that differ in number and functional characteristics from those in limb skeletal muscle. The EOMs also contain a large population of Pitx2-positive myogenic precursor cells that provide the EOMs with many of their unusual biological characteristics, such as myofiber remodeling and skeletal muscle disease sparing. This environment provides for rapid and efficient remodeling and regeneration after various types of injury. In addition, the EOMs show a remarkable ability to respond to perturbations of single muscles with coordinated changes in the other EOMs that move in the same plane. SUMMARY: These data will inform Ophthalmologists as they work toward developing new treatments for eye movement disorders, new approaches for repair after nerve or direct EOMs injury, as well as suggest potential explanations for the unusual disease propensity and disease sparing characteristics of human EOM.

Skeletal Muscle Tissue Clearing for LacZ and Fluorescent Reporters, and Immunofluorescence Staining.

Skeletal muscle is a highly ordered yet complex tissue containing several cell types that interact with each other in order to maintain structure and homeostasis. It is also a highly regenerative tissue that responds to damage in a highly intricate but stereotypic manner, with distinct spatial and temporal kinetics. Proper examination of this process requires one to look at the three-dimensional orientation of the cellular and subcellular components, which can be accomplished through tissue clearing. While there has been a recent surge of protocols to study biology in whole tissue, it has primarily focused on the nervous system. This chapter describes the workflow for whole mount analysis of murine skeletal muscle for LacZ reporters, fluorescent reporters and immunofluorescence staining. Using this technique, we are able to visualize LacZ reporters more effectively in deep tissue samples, and to perform fluorescent imaging with a depth greater than 1700 µm.

Acute failure of action potential conduction in mdx muscle reveals new mechanism of contraction-induced force loss.

A primary feature of skeletal muscle lacking the protein dystrophin, as occurring in Duchenne muscular dystrophy, is a hypersensitivity to contraction-induced strength loss. We tested the hypothesis that the extensive strength loss results from an impairment in the electrophysiological function of the plasmalemma specifically impaired action potential development. Anterior crural muscles from mdx and wildtype mice performed a single bout of 100 electrically stimulated eccentric contractions in vivo. Electromyography, specifically the M-wave, was analysed during muscle contraction to assess the ability of the tibialis anterior muscle plasmalemma to generate and conduct action potentials. During eccentric contractions, wildtype mice exhibited a 36% loss in torque about the ankle but mdx mice exhibited a greater torque loss of 73% (P < 0.001). Despite the loss of torque, there was no reduction in M-wave root mean square (RMS) for wildtype mice, which was in stark contrast to mdx mice that had a 55% reduction in M-wave RMS (P < 0.001). This impairment resolved within 24 h and coincided with a significant improvement in strength and membrane integrity. Intracellular measurements of resting membrane potential (RMP) in uninjured and injured extensor digitorum longus muscles were made to determine if a chronic depolarization had occurred, which could lead to impaired fibre excitability and/or altered action potential conduction properties. The distributions of RMP were not different between wildtype uninjured and injured muscle cells (median: -73.2 mV vs. -72.7 mV, P = 0.46) whereas there was a significant difference between mdx uninjured and injured cells (median: -71.5 mV vs. -56.6 mV, P < 0.001). These data show that mdx muscle fibres are depolarized after an injurious bout of eccentric contractions. These findings (i) suggest a major plasmalemma-based mechanism of strength loss underlying contraction-induced injury in Duchenne muscular dystrophy distinctly different from that for healthy muscle, and (ii) demonstrate dystrophin is critical for maintaining action potential generation and conduction after eccentric contractions.

Vascular-targeted therapies for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and an X-linked recessive, progressive muscle wasting disease caused by the absence of a functional dystrophin protein. Dystrophin has a structural role as a cytoskeletal stabilization protein and protects cells against contraction-induced damage. Dystrophin also serves a signaling role through mechanotransduction of forces and localization of neuronal nitric oxide synthase (nNOS), which produces nitric oxide (NO) to facilitate vasorelaxation. In DMD, the signaling defects produce inadequate tissue perfusion caused by functional ischemia due to a diminished ability to respond to shear stress induced endothelium-dependent dilation. Additionally, the structural defects seen in DMD render myocytes with an increased susceptibility to mechanical stress. The combination of both defects is necessary to generate myocyte damage, which induces successive rounds of myofiber degeneration and regeneration, loss of calcium homeostasis, chronic inflammatory response, fibrosis, and myonecrosis. In individuals with DMD, these processes inevitably cause loss of ambulation shortly after the first decade and an abbreviated life with death in the third or fourth decade due to cardio-respiratory anomalies. There is no known cure for DMD, and although the culpable gene has been identified for more than twenty years, research on treatments has produced few clinically relevant results. Several recent studies on novel DMD therapeutics are vascular targeted and focused on attenuating the inherent functional ischemia. One approach improves vasorelaxation capacity through pharmaceutical inhibition of either phosphodiesterase 5 (PDE5) or angiotensin-converting enzyme (ACE). Another approach increases the density of the underlying vascular network by inducing angiogenesis, and this has been accomplished through either direct delivery of vascular endothelial growth factor (VEGF) or by downregulating the VEGF decoy-receptor type 1 (VEGFR-1 or Flt-1). The pro-angiogenic approaches also seem to be pro-myogenic and could resolve the age-related decline in satellite cell (SC) quantity seen in mdx models through expansion of the SC juxtavascular niche. Here we review these four vascular targeted treatment strategies for DMD and discuss mechanisms, proof of concept, and the potential for clinical relevance associated with each therapy.

Improved regenerative myogenesis and muscular dystrophy in mice lacking Mkp5.

Duchenne muscular dystrophy (DMD) is a degenerative skeletal muscle disease caused by mutations in dystrophin. The degree of functional deterioration in muscle stem cells determines the severity of DMD. The mitogen-activated protein kinases (MAPKs), which are inactivated by MAPK phosphatases (MKPs), represent a central signaling node in the regulation of muscle stem cell function. Here we show that the dual-specificity protein phosphatase DUSP10/MKP-5 negatively regulates muscle stem cell function in mice. MKP-5 controlled JNK to coordinate muscle stem cell proliferation and p38 MAPK to control differentiation. Genetic loss of Mkp5 in mice improved regenerative myogenesis and dystrophin-deficient mdx mice lacking Mkp5 exhibited an attenuated dystrophic muscle phenotype. Hence, enhanced promyogenic MAPK activity preserved muscle stem cell function even in the absence of dystrophin and ultimately curtailed the pathogenesis associated with DMD. These results identify MKP-5 as an essential negative regulator of the promyogenic actions of the MAPKs and suggest that MKP-5 may serve as a target to promote muscle stem cell function in the treatment of degenerative skeletal muscle diseases.