ABSTRACT
Duchenne muscular dystrophy (DMD) is an intractable X-linked muscular dystrophy caused by mutations in the DMD gene. While many animal models have been used to study the disease, translating findings to humans has been challenging. Microminipigs, with their pronounced physiological similarity to humans and notably compact size amongst pig models, could offer a more representative model for human diseases. Here, we accomplished precise DMD modification in microminipigs by co-injecting embryos with Cas9 protein and a single-guide RNA targeting exon 23 of DMD. The DMD-edited microminipigs exhibited pronounced clinical phenotypes, including perturbed locomotion and body-wide skeletal muscle weakness and atrophy, alongside augmented serum creatine kinase levels. Muscle weakness was observed as of one month of age, respiratory and cardiac dysfunctions emerged by the sixth month, and the maximum lifespan was 29.9 months. Histopathological evaluations confirmed dystrophin deficiency and pronounced dystrophic pathology in the skeletal and myocardial tissues, demonstrating that these animals are an unprecedented model for studying human DMD. The model stands as a distinct and crucial tool in biomedical research, offering deep understanding of disease progression and enhancing therapeutic assessments, with potential to influence forthcoming treatment approaches.
Subject(s)
Disease Models, Animal , Dystrophin , Muscle, Skeletal , Muscular Dystrophy, Duchenne , Swine, Miniature , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/pathology , Muscular Dystrophy, Duchenne/physiopathology , Animals , Swine , Muscle, Skeletal/pathology , Muscle, Skeletal/metabolism , Dystrophin/genetics , Dystrophin/metabolism , Gene Editing , Humans , Male , PhenotypeABSTRACT
Muscular dystrophy in the NH-413 chicken is caused by a missense mutation in the WWP1 gene. WWP1 is a HECT-type E3 ubiquitin ligase containing four tandem WW domains that interact with proline-rich peptide motifs of target proteins, and a short region connecting the second and third WW domains is crucial for the E3 ligase to maintain an autoinhibitory state. A mutation of the arginine in the WW2-WW3 linker to glutamine is thought to affect WWP1 function, but there is little information on this mutation to date. In this study, we generated a transgenic (Tg) mouse model expressing the WWP1 transgene with the R436Q mutation, which corresponds to the missense mutation found in the NH-413 chicken. Tg mice showed marked degradation of mutant WWP1 proteins in various tissues, particularly in striated muscle. Immunoprecipitation analysis using a WWP1-specific antibody demonstrated that the mutant WWP1 proteins lacked the C-terminal catalytic cysteine residue that is required for their binding to the E2-substrate complex during their degradation. In vitro analysis using the R436Q mutant of WWP1 lacking this catalytic cysteine residue showed no autodegradation, indicating that the loss-of-function degradation of this protein is caused by self-ubiquitination. Tg mice expressing R436Q WWP1 did not show stunted growth or premature death. Furthermore, histological analysis did not reveal any obvious changes. These observations suggested that the R436Q mutant WWP1 protein, which is released from autoinhibitory mode by its missense mutation, does not have abnormally activated enzyme function to substrates before its self-degradation and loss of enzyme function.
Subject(s)
Mice, Transgenic , Mutation, Missense , Proteolysis , Ubiquitin-Protein Ligases , Ubiquitination , Animals , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Mutation, Missense/genetics , Mice , Humans , ChickensABSTRACT
Skeletal muscle can adjust to changes in physiological and pathological environments by regenerating using myogenic progenitor cells or adapting muscle fiber sizes and types, metabolism, and contraction ability. To study these changes, muscle samples should be appropriately prepared. Therefore, reliable techniques to accurately analyze and evaluate skeletal muscle phenotypes are required. However, although technical approaches to genetically investigating skeletal muscle are improving, the fundamental strategies for capturing muscle pathology are the same over the decades. Hematoxylin and eosin (H&E) staining or antibodies are the simplest and standard methodologies for assessing skeletal muscle phenotypes. In this chapter, we describe fundamental techniques and protocols for inducing skeletal muscle regeneration by using chemicals and cell transplantation, in addition to methods of preparing and evaluating skeletal muscle samples.
Subject(s)
Cell Transplantation , Muscle, Skeletal , Mice , Animals , Mice, Inbred mdx , Muscle, Skeletal/metabolism , Stem Cells/physiology , Dystrophin/geneticsABSTRACT
Duchenne muscular dystrophy (DMD) is a muscle disorder caused by DMD mutations and is characterized by neurobehavioural comorbidities due to dystrophin deficiency in the brain. The lack of Dp140, a dystrophin short isoform, is clinically associated with intellectual disability and autism spectrum disorders (ASDs), but its postnatal functional role is not well understood. To investigate synaptic function in the presence or absence of brain Dp140, we utilized two DMD mouse models, mdx23 and mdx52 mice, in which Dp140 is preserved or lacking, respectively. ASD-like behaviours were observed in pups and 8-week-old mdx52 mice lacking Dp140. Paired-pulse ratio of excitatory postsynaptic currents, glutamatergic vesicle number in basolateral amygdala neurons, and glutamatergic transmission in medial prefrontal cortex-basolateral amygdala projections were significantly reduced in mdx52 mice compared to those in wild-type and mdx23 mice. ASD-like behaviour and electrophysiological findings in mdx52 mice were ameliorated by restoration of Dp140 following intra-cerebroventricular injection of antisense oligonucleotide drug-induced exon 53 skipping or intra-basolateral amygdala administration of Dp140 mRNA-based drug. Our results implicate Dp140 in ASD-like behaviour via altered glutamatergic transmission in the basolateral amygdala of mdx52 mice.
Subject(s)
Dystrophin , Muscular Dystrophy, Duchenne , Animals , Brain/metabolism , Disease Models, Animal , Dystrophin/genetics , Dystrophin/metabolism , Exons , Mice , Muscular Dystrophy, Duchenne/genetics , Social BehaviorABSTRACT
Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive muscular weakness because of the loss of dystrophin. Extracellular Ca2+ flows into the cytoplasm through membrane tears in dystrophin-deficient myofibers, which leads to muscle contracture and necrosis. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) takes up cytosolic Ca2+ into the sarcoplasmic reticulum, but its activity is decreased in dystrophic muscle. Here, we show that an allosteric SERCA activator, CDN1163, ameliorates dystrophic phenotypes in dystrophin-deficient mdx mice. The administration of CDN1163 prevented exercise-induced muscular damage and restored mitochondrial function. In addition, treatment with CDN1163 for 7 weeks enhanced muscular strength and reduced muscular degeneration and fibrosis in mdx mice. Our findings provide preclinical proof-of-concept evidence that pharmacological activation of SERCA could be a promising therapeutic strategy for DMD. Moreover, CDN1163 improved muscular strength surprisingly in wild-type mice, which may pave the new way for the treatment of muscular dysfunction.
Subject(s)
Dystrophin/genetics , Muscular Dystrophy, Duchenne/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Animals , Calcium/metabolism , Disease Models, Animal , Dystrophin/deficiency , Humans , Mice , Mice, Inbred mdx , Muscle Contraction/genetics , Muscle Weakness/genetics , Muscle Weakness/pathology , Muscular Atrophy/genetics , Muscular Atrophy/pathology , Muscular Dystrophy, Duchenne/pathology , Phenotype , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum/pathologyABSTRACT
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMDJ) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.
Subject(s)
Cyclin-Dependent Kinase 4/genetics , Dystrophin/genetics , Morpholinos/pharmacology , Muscular Dystrophy, Duchenne/therapy , Telomerase/genetics , Animals , Cell Line , Dogs , Exons/genetics , Genetic Therapy , Humans , Mice , Morpholinos/genetics , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/pathology , Myoblasts/metabolism , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , Peptides/genetics , Peptides/pharmacology , RNA Splice Sites/geneticsABSTRACT
Temple and Kagami-Ogata syndromes are genomic imprinting diseases caused by maternal and paternal duplication of human chromosome 14, respectively. They exhibit different postnatal muscle-related symptoms as well as prenatal placental problems. Using the mouse models for these syndromes, it has been demonstrated that retrotransposon gag like 1 [Rtl1, also known as paternally expressed 11 (Peg11)] located in the mouse orthologous imprinted region is responsible for the prenatal placental problems because it is an essential placental gene for maintenance of fetal capillary network during gestation. However, the causative imprinted gene for the postnatal muscle-related symptoms remains unknown. Here, we demonstrate that Rtl1 also plays an important role in fetal/neonatal skeletal muscle development: its deletion and overproduction in mice lead to neonatal lethality associated with severe but distinct skeletal muscle defects, similar to those of Temple and Kagami-Ogata syndromes, respectively. Thus, it is strongly suggested that RTL1 is the major gene responsible for the muscle defects in addition to the placental defects in these two genomic imprinting diseases. This is the first example of an LTR retrotransposon-derived gene specific to eutherians contributing to eutherian skeletal muscle development.
Subject(s)
Abnormalities, Multiple/metabolism , Abnormalities, Multiple/pathology , Muscles/abnormalities , Pregnancy Proteins/deficiency , Animals , Animals, Newborn , Cell Differentiation , Cell Proliferation , Desmin/metabolism , Female , Fetus/metabolism , Gene Expression Regulation, Developmental , Humans , Male , Mice, Inbred C57BL , Mice, Knockout , Models, Genetic , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Muscles/embryology , Muscles/pathology , Mutation/genetics , Pregnancy Proteins/genetics , Pregnancy Proteins/metabolism , Satellite Cells, Skeletal Muscle/metabolism , Syndrome , Time FactorsABSTRACT
The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (VD3), exerts its tissue-specific actions through binding to its intracellular vitamin D receptor (VDR) which functions as a heterodimer with retinoid X receptor (RXR) to recognize vitamin D response elements (VDRE) and activate target genes. Upregulation of VDR in murine skeletal muscle cells occurs concomitantly with transcriptional regulation of key myogenic factors upon VD3 administration, reinforcing the notion that VD3 exerts beneficial effects on muscle. Herein we elucidated the regulatory role of VD3/VDR axis on the expression of dystrobrevin alpha (DTNA), a member of dystrophin-associated protein complex (DAPC). In C2C12 cells, Dtna and VDR gene and protein expression were upregulated by 1-50 nM of VD3 during all stages of myogenic differentiation. In the dystrophic-derived H2K-mdx52 cells, upregulation of DTNA by VD3 occurred upon co-transfection of VDR and RXR expression vectors. Silencing of MyoD1, an E-box binding myogenic transcription factor, did not alter the VD3-mediated Dtna induction, but Vdr silencing abolished this effect. We also demonstrated that VD3 administration enhanced the muscle-specific Dtna promoter activity in presence of VDR/RXR only. Through site-directed mutagenesis and chromatin immunoprecipitation assays, we have validated a VDRE site in Dtna promoter in myogenic cells. We have thus proved that the positive regulation of Dtna by VD3 observed during in vitro murine myogenic differentiation is VDR mediated and specific. The current study reveals a novel mechanism of VDR-mediated regulation for Dtna, which may be positively explored in treatments aiming to stabilize the DAPC in musculoskeletal diseases.
Subject(s)
Dystrophin-Associated Proteins/genetics , Muscles/metabolism , Neuropeptides/genetics , Receptors, Calcitriol/physiology , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Female , Gene Expression Regulation/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscles/drug effects , Muscles/physiology , Myoblasts, Skeletal/drug effects , Myoblasts, Skeletal/physiology , Transcriptional Activation/drug effects , Vitamin D/analogs & derivatives , Vitamin D/pharmacology , Vitamin D Response Element/drug effects , Vitamin D Response Element/geneticsABSTRACT
Diabetes mellitus is associated with various disorders of the locomotor system including the decline in mass and function of skeletal muscle. The mechanism underlying this association has remained ambiguous, however. We now show that the abundance of the transcription factor KLF15 as well as the expression of genes related to muscle atrophy are increased in skeletal muscle of diabetic model mice, and that mice with muscle-specific KLF15 deficiency are protected from the diabetes-induced decline of skeletal muscle mass. Hyperglycemia was found to upregulate the KLF15 protein in skeletal muscle of diabetic animals, which is achieved via downregulation of the E3 ubiquitin ligase WWP1 and consequent suppression of the ubiquitin-dependent degradation of KLF15. Our results revealed that hyperglycemia, a central disorder in diabetes, promotes muscle atrophy via a WWP1/KLF15 pathway. This pathway may serve as a therapeutic target for decline in skeletal muscle mass accompanied by diabetes mellitus.
Subject(s)
Diabetes Mellitus, Experimental/complications , Hyperglycemia/complications , Kruppel-Like Transcription Factors/metabolism , Muscle, Skeletal/pathology , Muscular Atrophy/pathology , Ubiquitin-Protein Ligases/metabolism , Animals , Benzhydryl Compounds/administration & dosage , COS Cells , Chlorocebus aethiops , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/drug therapy , Down-Regulation , Female , Gene Expression Profiling , Glucosides/administration & dosage , HEK293 Cells , Humans , Hyperglycemia/blood , Hyperglycemia/chemically induced , Hyperglycemia/drug therapy , Kruppel-Like Transcription Factors/genetics , Male , Mice , Mice, Knockout , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscular Atrophy/etiology , Muscular Atrophy/genetics , Muscular Atrophy/prevention & control , Proteolysis , Signal Transduction/genetics , Sodium-Glucose Transporter 2 Inhibitors/administration & dosage , Streptozocin/toxicity , Up-RegulationABSTRACT
MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at the post-transcriptional level. Several miRNAs are exclusively expressed in skeletal muscle and participate in the regulation of muscle differentiation by interacting with myogenic factors. These miRNAs can be found at high levels in the serum of patients and animal models for Duchenne muscular dystrophy, which is expected to be useful as biomarkers for their clinical conditions. By miRNA microarray analysis, we identified miR-188 as a novel miRNA that is elevated in the serum of the muscular dystrophy dog model, CXMDJ. miR-188 was not muscle-specific miRNA, but its expression was up-regulated in skeletal muscles associated with muscle regeneration induced by cardiotoxin-injection in normal dogs and mice. Manipulation of miR-188 expression using antisense oligo and mimic oligo RNAs alters the mRNA expression of the myogenic regulatory factors, MRF4 and MEF2C. Our results suggest that miR-188 is a new player that participates in the gene regulation process of muscle differentiation and that it may serve as a serum biomarker reflecting skeletal muscle regeneration.
Subject(s)
Biomarkers/blood , Gene Expression Regulation , MicroRNAs/genetics , Muscle, Skeletal/metabolism , Muscular Dystrophy, Animal/genetics , Muscular Dystrophy, Duchenne/genetics , Animals , Cell Differentiation , Cells, Cultured , Disease Models, Animal , Dogs , Mice , Muscle, Skeletal/pathology , Muscular Dystrophy, Animal/blood , Muscular Dystrophy, Animal/pathology , Muscular Dystrophy, Duchenne/blood , Muscular Dystrophy, Duchenne/pathology , Myoblasts/cytology , Myoblasts/metabolismABSTRACT
Duchenne muscular dystrophy is a lethal X-linked muscle disorder. We have already reported that osteopontin (OPN), an inflammatory cytokine and myogenic factor, is expressed in the early dystrophic phase in canine X-linked muscular dystrophy in Japan, a dystrophic dog model. To further explore the possibility of OPN as a new biomarker for disease activity in Duchenne muscular dystrophy, we monitored serum OPN levels in dystrophic and wild-type dogs at different ages and compared the levels to other serum markers, such as serum creatine kinase, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1. Serum OPN levels in the dystrophic dogs were significantly elevated compared with those in wild-type dogs before and 1 hour after a cesarean section birth and at the age of 3 months. The serum OPN level was significantly correlated with the phenotypic severity of dystrophic dogs at the period corresponding to the onset of muscle weakness, whereas other serum markers including creatine kinase were not. Immunohistologically, OPN was up-regulated in infiltrating macrophages and developmental myosin heavy chain-positive regenerating muscle fibers in the dystrophic dogs, whereas serum OPN was highly elevated. OPN expression was also observed during the synergic muscle regeneration process induced by cardiotoxin injection. In conclusion, OPN is a promising biomarker for muscle regeneration in dystrophic dogs and can be applicable to boys with Duchenne muscular dystrophy.
Subject(s)
Muscle, Skeletal/physiology , Muscular Dystrophy, Duchenne/physiopathology , Osteopontin/metabolism , Regeneration/physiology , Age Factors , Animals , Biomarkers/metabolism , Cobra Cardiotoxin Proteins/toxicity , Diaphragm/metabolism , Dogs , Male , Matrix Metalloproteinase 9/metabolism , Muscle, Skeletal/metabolism , Muscular Dystrophy, Animal/physiopathology , Phenotype , Tissue Inhibitor of Metalloproteinase-1/metabolismABSTRACT
A missense mutation in the gene encoding WWP1 was identified as the most promising candidate responsible for chicken muscular dystrophy (MD) by genetic linkage analysis. WWP1 is a HECT-type E3 ubiquitin protein ligase composed of 922 amino acids, which contains 4 tandem WW domains that interact with the proline-rich peptide motifs of target proteins. The missense mutation changes arginine 441 that is located in the centre of the WW domains into glutamine (R441Q), which potentially affects the function of the WWP1 protein. Here, we show that WWP1 is detected as â¼130-kDa protein that localizes to various structures, such as the plasma membrane (sarcolemma), sarcoplasmic reticulum, mitochondria and nucleus, in normal chicken skeletal muscle. However, in MD chickens, the mutant WWP1 protein was markedly degraded and was absent in the sarcolemma. These changes were also observed in the muscles of chickens in early pre-pathological states. Moreover, in vitro expression analysis showed significant degradation of mutant, but not wild-type WWP1, specifically in myogenic cells. Altogether, our data revealed that the R441Q missense mutation in the WWP1 protein causes degradation and loss of the sarcolemmal localization of WWP1, which may play a role in the pathogenesis of chicken MD.
Subject(s)
Chickens/metabolism , Muscle, Skeletal/metabolism , Muscular Dystrophy, Animal/metabolism , Poultry Diseases/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Nucleus/metabolism , Chickens/genetics , Immunohistochemistry , Mitochondria/metabolism , Muscle, Skeletal/pathology , Muscular Dystrophy, Animal/genetics , Mutation, Missense , Poultry Diseases/genetics , Proteolysis , Sarcolemma/metabolism , Sarcoplasmic Reticulum/metabolism , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
Matrix metalloprotease (MMP)-9 is an endopeptidase associated with the pathogenesis of Duchenne muscular dystrophy (DMD). The precise function of MMP-9 in DMD has not been elucidated to date. We investigated the effect of genetic ablation of MMP-9 in the mdx mouse model (mdx/Mmp9(-/-)). At the early disease stage, the muscles of mdx/Mmp9(-/-) mice showed reduced necrosis and neutrophil invasion, accompanied by down-regulation of chemokine MIP-2. In addition, muscle regeneration was enhanced, which coincided with increased macrophage infiltration and upregulation of MCP-1, and resulted in increased muscle strength. The mdx/Mmp9(-/-) mice also displayed accelerated upregulation of osteopontin expression in skeletal muscle at the acute onset phase of dystrophy. However, at a later disease stage, the mice exhibited muscle growth impairment through altered expression of myogenic factors, and increased fibroadipose tissue. These results showed that MMP-9 might have multiple functions during disease progression. Therapy targeting MMP-9 may improve muscle pathology and function at the early disease stage, but continuous inhibition of this protein may result in the accumulation of fibroadipose tissues and reduced muscle strength at the late disease stage.
ABSTRACT
Cell polarity depends on extrinsic spatial cues and intrinsic polarity proteins including PAR-aPKC proteins. In mammalian epithelial cells, cell-cell contacts provide spatial cues that activate the aPKC-PAR-3-PAR-6 complex to establish the landmark of the initial cellular asymmetry. PAR-1, a downstream target of the aPKC-PAR-3-PAR-6 complex, mediates further development of the apical and basolateral membrane domains. However, the relationships between the PAR-aPKC proteins and other extrinsic spatial cues provided by the extracellular matrix (ECM) remain unclear. Here, we show that PAR-1 colocalizes with laminin receptors and is required for the assembly of extracellular laminin on the basal surface of epithelial cells. Furthermore, PAR-1 regulates the basolateral localization of the dystroglycan (DG) complex, one of the laminin receptors essential for basement membrane formation. We also show that PAR-1 interacts with the DG complex and is required for the formation of a functional DG complex. These results reveal the presence of a novel inside-out pathway in which an intracellular polarity protein regulates the ECM organization required for epithelial cell polarity and tissue morphogenesis.
Subject(s)
Cell Polarity/physiology , Dystroglycans/metabolism , Laminin/physiology , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Membrane/metabolism , Cells, Cultured , Dogs , Extracellular Matrix/metabolism , Microscopy, Fluorescence , Receptors, Laminin/metabolism , TransfectionABSTRACT
Autosomal recessive limb-girdle muscular dystrophy type 2D (LGMD 2D) is caused by mutations in the alpha-sarcoglycan gene (alpha-SG). The absence of alpha-SG results in the loss of the SG complex at the sarcolemma and compromises the integrity of the sarcolemma. To establish a method for recombinant adeno-associated virus (rAAV)-mediated alpha-SG gene therapy into alpha-SG-deficient muscle, we constructed rAAV serotypes 2 and 8 expressing the human alpha-SG gene under the control of the ubiquitous cytomegalovirus promoter (rAAV2-alpha-SG and rAAV8-alpha-SG). We compared the transduction profiles and evaluated the therapeutic effects of a single intramuscular injection of rAAVs into alpha-SG-deficient (Sgca(-/-)) mice. Four weeks after rAAV2 injection into the tibialis anterior (TA) muscle of 10-day-old Sgca(-/-) mice, transduction of the alpha-SG gene was localized to a limited area of the TA muscle. On the other hand, rAAV8-mediated alpha-SG expression was widely distributed in the hind limb muscle, and persisted for 7 months without inducing cytotoxic and immunological reactions, with a reversal of the muscle pathology and improvement in the contractile force of the Sgca(-/-) muscle. This extensive rAAV8-mediated alpha-SG transduction in LGMD 2D model animals paves the way for future clinical application.
Subject(s)
Dependovirus/genetics , Genetic Therapy/methods , Muscle, Skeletal/virology , Muscular Dystrophies, Limb-Girdle/therapy , Recombination, Genetic , Sarcoglycans/metabolism , Transduction, Genetic , Animals , Dependovirus/classification , Genetic Vectors , Humans , Mice , Mice, Knockout , Muscle Contraction/physiology , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Sarcoglycans/deficiency , Sarcoglycans/genetics , Serotyping , Treatment OutcomeABSTRACT
In skeletal muscles, the sarcolemma is possibly stabilized and protected against contraction-imposed stress by intermediate filaments (IFs) tethered to costameric sarcolemma. Although there is emerging evidence that plectin links IFs to costameres through dystrophin-glycoprotein complexes (DGC), the molecular organization from plectin to costameres still remains unclear. Here, we show that plectin 1, a plectin isoform expressed in skeletal muscle, can interact with beta-synemin, actin and a DGC component, alpha-dystrobrevin, in vitro. Ultrastructurally, beta-synemin molecules appear to be incorporated into costameric dense plaques, where they seem to serve as actin-associated proteins rather than IF proteins. In fact, they can bind actin and alpha-dystrobrevin in vitro. Moreover, in vivo immunoprecipitation analyses demonstrated that beta-synemin- and plectin-immune complexes from lysates of muscle light microsomes contained alpha-dystrobrevin, dystrophin, nonmuscle actin, metavinculin, plectin and beta-synemin. These findings suggest a model in which plectin 1 interacts with DGC and integrin complexes directly, or indirectly through nonmuscle actin and beta-synemin within costameres. The DGC and integrin complexes would cooperate to stabilize and fortify the sarcolemma by linking the basement membrane to IFs through plectin 1, beta-synemin and actin. Besides, the two complexes, together with plectin and IFs, might have their own functions as platforms for distinct signal transduction.
Subject(s)
Actins/metabolism , Dystrophin-Associated Proteins/metabolism , Intermediate Filament Proteins/metabolism , Plectin/metabolism , Actins/genetics , Animals , Dystrophin/deficiency , Dystrophin/metabolism , Dystrophin-Associated Proteins/genetics , Intermediate Filament Proteins/genetics , Intermediate Filaments/metabolism , Intermediate Filaments/ultrastructure , Mice , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/ultrastructure , Muscular Dystrophy, Animal/genetics , Muscular Dystrophy, Animal/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Myoblasts, Skeletal/cytology , Myoblasts, Skeletal/metabolism , Plectin/chemistry , Plectin/genetics , Protein Binding , Rats , Sarcolemma/metabolism , Sarcolemma/ultrastructureABSTRACT
The sarcoglycans (SGs), transmembrane components of the dystrophin-associated glycoprotein complex, are stable and functional only when they assemble into a tetrameric complex in muscle cells. A defect in any one of the four SG members disrupts the entire SG complex (SGC) and causes limb-girdle muscular dystrophy. zeta-SG has been recently found as a transmembrane protein homologous to gamma-SG and delta-SG. To characterize zeta-SG in complex formation, we co-transfected expression vectors encoding all six SGs (alpha-, beta-, gamma-, delta-, epsilon- and zeta-SG) and dystroglycan into Chinese hamster ovary cells. Immunoprecipitation analysis showed that zeta-SG or gamma-SG formed a SGC with beta-SG and delta-SG plus alpha-SG or epsilon-SG, revealing that zeta-SG can form two types of SGCs (alpha-beta-zeta-delta or epsilon-beta-zeta-delta). This result indicates the functional resemblance of zeta-SG to gamma-SG rather than delta-SG, although phylogenetic analysis suggests that zeta-SG is evolutionally closer to delta-SG than to gamma-SG. Reverse transcription (RT)-PCR showed that the expression pattern of the transcript was almost the reciprocal of that of gamma-SG in various mouse tissues and that the zeta-SG transcript was especially abundant in the brain, suggesting that zeta-SG might play a particular role in the central nervous system.
Subject(s)
Macromolecular Substances/metabolism , Sarcoglycans/classification , Sarcoglycans/physiology , Structural Homology, Protein , Animals , Brain/metabolism , CHO Cells , Cell Line , Cell Membrane/chemistry , Cell Membrane/metabolism , Cricetinae , Mice , Models, Biological , Phylogeny , Sarcoglycans/analysis , Sarcoglycans/geneticsABSTRACT
Dystrophin and the dystrophin-associated protein (DAP) complex protect the sarcolemma against contraction-induced injury and serve as a mechanical link between the extracellular matrix and the actin cytoskeleton. Some of the functional properties of the DAP complex are mediated by its sarcoglycan (SG) subcomplex, which is composed of alpha-, beta-, gamma- and delta-SGs. Autosomal recessive limb-girdle muscular dystrophy type-2D (LGMD 2D) results from reduction in SG subcomplex levels caused by specific mutations in the muscle-specific alpha-SG gene. epsilon-SG is a widely expressed homolog of the muscle-specific alpha-SG, and expression of epsilon-SG may compensate for the pathologic changes in alpha-SG function. Thus, the goal of the present study was to investigate whether overexpression of epsilon-SG can compensate for dysfunction of alpha-SG. Several transgenic mouse lines that overexpress epsilon-SG in skeletal muscle were established. Overexpression of epsilon-SG in normal mice resulted in substitution of epsilon-SG for alpha-SG in the SG complex of skeletal muscle without any obvious abnormalities. To determine whether an increase in epsilon-SG expression may prevent muscular dystrophy in the context of alpha-SG-deficiency, these epsilon-SG transgenic mice were crossed with alpha-SG deficient mice. alpha-SG-deficient mice overexpressing epsilon-SG exhibited no skeletal muscle cell membrane damage or abnormal contraction. These data suggest that the overexpression of epsilon-SG may represent a therapeutic strategy for treatment of LGMD 2D.
Subject(s)
Muscle, Skeletal/metabolism , Muscular Dystrophies, Limb-Girdle/metabolism , Sarcoglycans/metabolism , Animals , Disease Models, Animal , Dystrophin/metabolism , Dystrophin-Associated Proteins , Gene Expression , Mice , Mice, Transgenic , Muscular Dystrophies, Limb-Girdle/genetics , Sarcoglycans/geneticsABSTRACT
Mutations in the dysferlin gene underlie two phenotypically distinct muscular dystrophies: Miyoshi myopathy and limb-girdle muscular dystrophy 2B. Dysferlin was proposed to have a putative functional role in mediating the fusion of intracellular vesicles to the sarcolemma during injury-induced membrane repair, but dysferlin has been found not only at the sarcolemma but also within the cytoplasm of skeletal muscle fibers by immunohistochemistry. In this study, we examined the subcellular localization of dysferlin in skeletal muscle by immunohistochemical and biochemical analyses to elucidate other functional roles of dysferlin. Immunohistochemistry confirmed granular cytoplasmic expression pattern of dysferlin in muscle fibers. Subcellular membrane fractionation revealed that a portion of dysferlin associated with a T-tubule-enriched intracellular membrane fraction as well as a sarcolemmal fraction. This indication was consistent with subsequent results that dysferlin coprecipitates by immunoprecipitation with the dihydropyridine receptor (DHPR), a protein complex localized in T-tubules. Moreover, both proteins were observed to partially colocalize by double immunofluorescent labeling in skeletal muscle fibers. We also found that caveolin-3, previously shown to interact with dysferlin, coprecipitates with DHPR. These results demonstrated that dysferlin may be involved in the formation of an oligomeric complex with DHPR and caveolin-3. Caveolin-3 has been also reported to participate in an insulin-regulated transport mechanism in muscle, and caveolin-3-containing vesicles might traffic between intracellular sites and target sites on the sarcolemma and T-tubules. Therefore, it is very intriguing to assume that dysferlin might be involved in the fusion of caveolin-3-containing vesicles with T-tubules.
Subject(s)
Calcium Channels, L-Type/metabolism , Membrane Proteins/metabolism , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Animals , Carrier Proteins/metabolism , Caveolin 3/metabolism , Cytoplasm/metabolism , Dysferlin , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Immunohistochemistry , In Vitro Techniques , Mice , Mice, Inbred BALB C , Microsomes/metabolism , Rats , Rats, Wistar , Sarcolemma/ultrastructureABSTRACT
BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by the absence of the muscle cytoskeletal protein dystrophin. Utrophin is an autosomal homologue of dystrophin, and overexpression of the protein is expected to compensate for the defect of dystrophin. The utrophin gene has two promoters, A and B, and promoter A of the utrophin gene is a possible target of pharmacological interventions for DMD because A-utrophin is up-regulated in dystrophin-deficient mdx skeletal and cardiac muscles. To investigate the utrophin promoter A activity in vivo, we generated nuclear localization signal-tagged LacZ transgenic mice, where the LacZ gene was driven by the 5-kb flanking region of the A-utrophin gene. METHODS: Four transgenic lines were established by mating four independent founders with C57BL/6J mice. The levels of mRNA for beta-galactosidase in several tissues were examined by RT-PCR. Cryosections from several tissues were stained with hematoxylin and eosin (H&E) and with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). RESULTS: The 5-kb upstream region of the A-utrophin gene showed high transcriptional activity in liver, testis, colon, submandibular gland, and small intestine, consistent with the endogenous expression of utrophin protein. Surprisingly, the levels of both beta-gal protein and mRNA for the transgene in cardiac and skeletal muscles were extremely low, even in nuclei near the neuromuscular junctions. These results indicate that the regulation of the utrophin gene in striated muscle is different from that in non-muscle tissues. CONCLUSIONS: Our results clearly showed that the utrophin A promoter is not sufficient to drive expression in muscle, but other regulatory elements are required.