Development Of Dystrophy Research Articles

Development of muscular dystrophy in a CRISPR-engineered mutant rabbit model with frame-disrupting ANO5 mutations

Limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi myopathy type 3 (MMD3) are autosomal recessive muscular dystrophy caused by mutations in the gene encoding anoctamin-5 (ANO5), which belongs to the anoctamin protein family. Two independent lines of mice with complete disruption of ANO5 transcripts did not exhibit overt muscular dystrophy phenotypes; instead, one of these mice was observed to present with some abnormality in sperm motility. In contrast, a third line of ANO5-knockout (KO) mice with residual expression of truncated ANO5 expression was reported to display defective membrane repair and very mild muscle pathology. Many of the ANO5-related patients carry point mutations or small insertions/deletions (indels) in the ANO5 gene. To more closely mimic the human ANO5 mutations, we engineered mutant ANO5 rabbits via co-injection of Cas9 mRNA and sgRNA into the zygotes. CRISPR-mediated small indels in the exon 12 and/or 13 in the mutant rabbits lead to the development of typical signs of muscular dystrophy with increased serum creatine kinase (CK), muscle necrosis, regeneration, fatty replacement and fibrosis. This novel ANO5 mutant rabbit model would be useful in studying the disease pathogenesis and therapeutic treatments for ANO5-deficient muscular dystrophy.

PKC Proteins and Muscular Dystrophy

Protein Kinase Cs (PKCs) are a family of 10 isoenzymes with critical roles in cell physiological processes like proliferation, differentiation, apoptosis. Muscular dystrophies are a heterogenous group of genetic degenerative diseases that affect skeletal and cardiac muscles. In the development of muscular dystrophies, several transduction pathways have been studied. A possible link between muscular dystrophies and PKCs have been recently proposed. After a brief description of the possible transduction pathways that are involved in the development of these genetic diseases, we summarize recent evidence on the role of PKC proteins in muscular dystrophies, with the aim to review possible candidates in molecular therapy of these pathologies.

COUP-TFII regulates satellite cell function and muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle-wasting disease caused by mutations in the dystrophin gene. Although dystrophin deficiency in myofiber triggers the disease's pathological changes, the degree of satellite cell (SC) dysfunction defines disease progression. Here, we have identified chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) hyperactivity as a contributing factor underlying muscular dystrophy in a dystrophin-deficient murine model of DMD. Ectopic expression of COUP-TFII in murine SCs led to Duchenne-like dystrophy in the muscles of control animals and exacerbated degenerative myopathies in dystrophin-deficient mice. COUP-TFII-overexpressing mice exhibited regenerative failure that was attributed to deficient SC proliferation and myoblast fusion. Mechanistically, we determined that COUP-TFII coordinated a regenerative program through combined regulation of multiple promyogenic factors. Furthermore, inhibition of COUP-TFII preserved SC function and counteracted the muscle weakness associated with Duchenne-like dystrophy in the murine model, suggesting that targeting COUP-TFII is a potential treatment for DMD. Together, our findings reveal a regulatory role of COUP-TFII in the development of muscular dystrophy and open up a potential therapeutic opportunity for managing disease progression in patients with DMD.

Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition.

Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.

Absence of Dystrophin Disrupts Skeletal Muscle Signaling: Roles of Ca2+, Reactive Oxygen Species, and Nitric Oxide in the Development of Muscular Dystrophy.

Dystrophin is a long rod-shaped protein that connects the subsarcolemmal cytoskeleton to a complex of proteins in the surface membrane (dystrophin protein complex, DPC), with further connections via laminin to other extracellular matrix proteins. Initially considered a structural complex that protected the sarcolemma from mechanical damage, the DPC is now known to serve as a scaffold for numerous signaling proteins. Absence or reduced expression of dystrophin or many of the DPC components cause the muscular dystrophies, a group of inherited diseases in which repeated bouts of muscle damage lead to atrophy and fibrosis, and eventually muscle degeneration. The normal function of dystrophin is poorly defined. In its absence a complex series of changes occur with multiple muscle proteins showing reduced or increased expression or being modified in various ways. In this review, we will consider the various proteins whose expression and function is changed in muscular dystrophies, focusing on Ca(2+)-permeable channels, nitric oxide synthase, NADPH oxidase, and caveolins. Excessive Ca(2+) entry, increased membrane permeability, disordered caveolar function, and increased levels of reactive oxygen species are early changes in the disease, and the hypotheses for these phenomena will be critically considered. The aim of the review is to define the early damage pathways in muscular dystrophy which might be appropriate targets for therapy designed to minimize the muscle degeneration and slow the progression of the disease.

Genetic Engineering of Dystroglycan in Animal Models of Muscular Dystrophy.

In skeletal muscle, dystroglycan (DG) is the central component of the dystrophin-glycoprotein complex (DGC), a multimeric protein complex that ensures a strong mechanical link between the extracellular matrix and the cytoskeleton. Several muscular dystrophies arise from mutations hitting most of the components of the DGC. Mutations within the DG gene (DAG1) have been recently associated with two forms of muscular dystrophy, one displaying a milder and one a more severe phenotype. This review focuses specifically on the animal (murine and others) model systems that have been developed with the aim of directly engineering DAG1 in order to study the DG function in skeletal muscle as well as in other tissues. In the last years, conditional animal models overcoming the embryonic lethality of the DG knock-out in mouse have been generated and helped clarifying the crucial role of DG in skeletal muscle, while an increasing number of studies on knock-in mice are aimed at understanding the contribution of single amino acids to the stability of DG and to the possible development of muscular dystrophy.

Facioscapulohumeral muscular dystrophy region gene 1 over-expression causes primary defects of myogenic stem cells

Overexpression of facioscapulohumeral muscular dystrophy region gene 1 (FRG1) in mice, frogs and worms leads to muscular and vascular abnormalities. Nevertheless, the mechanism that follows FRG1 overexpression and finally leads to muscular defects is currently unknown. Here, we show that the earliest phenotype displayed by mice overexpressing FRG1 is a postnatal muscle-growth defect. Long before the development of muscular dystrophy, FRG1 mice also exhibit a muscle regeneration impairment. Ex vivo and in vivo experiments revealed that FRG1 overexpression causes myogenic stem cell activation and proliferative, clonogenic and differentiation defects. A comparative gene expression profiling of muscles from young pre-dystrophic wild-type and FRG1 mice identified differentially expressed genes in several gene categories and networks that could explain the emerging tissue and myogenic stem cell defects. Overall, our study provides new insights into the pathways regulated by FRG1 and suggests that muscle stem cell defects could contribute to the pathology of FRG1 mice.

Caveolae and non-caveolae lipid raft microdomains of human umbilical vein endothelial cells contain utrophin-associated protein complexes

Several studies have shown the importance of dystrophin-associated protein complex in the development of muscular dystrophies and dilated cardiomyopathy associated to vascular dysfunction. In vascular endothelium, dystrophin is substituted for utrophin (autosomal homolog of dystrophin); however, its role in this tissue is unknown. Therefore, it is important to obtain a more extensive knowledge of utrophin and its associated proteins in endothelial cells. In a previous study, we demonstrated the presence of utrophin-associated protein complex (UAPC) in human umbilical vein endothelial cells HUVEC, which interacts with caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS). Also, some of our observations suggested the presence of this complex in distinct membrane domains. Therefore, the aim of this study was to analyze the presence of the UAPC in caveolae and non-caveolae lipid rafts domains of HUVEC at baseline and with a mechanical stimulus. It was demonstrated, by subcellular fractionation and co-immunoprecipitation assays, the association of UAPC with Cav-1 and eNOS in caveolae domains, as well as its interaction with eNOS in non-caveolae lipid raft domains. Additionally, it was also observed that mechanical stress on endothelial cells induced activation and release of eNOS from both caveolae and non-caveolae lipid raft associated to UAPC. Together these results suggest that UAPC located in caveolae and non-caveolae lipid raft domains of HUVECs may have a mechanosensory function that could participate in the control of eNOS activity.

Stress and muscular dystrophy: A genetic screen for Dystroglycan and Dystrophin interactors in Drosophila identifies cellular stress response components

In Drosophila, like in humans, Dystrophin Glycoprotein Complex (DGC) deficiencies cause a life span shortening disease, associated with muscle dysfunction. We performed the first in vivo genetic interaction screen in ageing dystrophic muscles and identified genes that have not been shown before to have a role in the development of muscular dystrophy and interact with dystrophin and/or dystroglycan. Mutations in many of the found interacting genes cause age-dependent morphological and heat-induced physiological defects in muscles, suggesting their importance in the tissue. Majority of them is phylogenetically conserved and implicated in human disorders, mainly tumors and myopathies. Functionally they can be divided into three main categories: proteins involved in communication between muscle and neuron, and interestingly, in mechanical and cellular stress response pathways. Our data show that stress induces muscle degeneration and accelerates age-dependent muscular dystrophy. Dystrophic muscles are already compromised; and as a consequence they are less adaptive and more sensitive to energetic stress and to changes in the ambient temperature. However, only dystroglycan, but not dystrophin deficiency causes extreme myodegeneration induced by energetic stress suggesting that dystroglycan might be a component of the low-energy pathway and act as a transducer of energetic stress in normal and dystrophic muscles.

The relationship between serum levels of vascular endothelial growth factor and muscular dystrophy development

Objective To detect serum levels of vascular endothelial growth factor (VEGF) in patients with muscular dystrophy and further to examine whether VEGF is associated with muscular dystrophy development. Methods We measured serum levels of VEGF in 32 patients with DMD,9 with Becker muscular dystrophy (BMD),5 with myotonic dystrophy (DM),as well as in 15 healthy and eight disease controls. Results The serum level of VEGF in the DMD patients was (274.7±2.52)pg/ml,while it was(358.8±9.64) pg/ml in the BMD patients, (165.0±6.34) pg/ml in the DM patients, (1 48.3±2.91) pg/ml in the healthy controls,and (153.7±5.42) pg/ml in the disease controls. The level of VEGF in BMD was significantly elevated, as compared with DM and control groups. Further, the level of VEGF in the bedridden sub-group of DMD patients was significantly elevated as compared with chair-bound DMD, DM, and control groups. Conclusion We concluded that VEGF may reflect hypoxic and/or ischemic conditions in muscle tissue, and have a relationship with the process of disease progression in DMD and BMD patients. Key words: Becker muscular dystrophy (BMD); Duchenne muscular dystrophy (DMD); Vascular endothelial growth factor (VEGF)

Transgenic Overexpression of ADAM12 Suppresses Muscle Regeneration and Aggravates Dystrophy in Aged mdx Mice

Muscular dystrophies are characterized by insufficient restoration and gradual replacement of the skeletal muscle by fat and connective tissue. ADAM12 has previously been shown to alleviate the pathology of young dystrophin-deficient mdx mice, a model for Duchenne muscular dystrophy. The observed effect of ADAM12 was suggested to be mediated via a membrane-stabilizing up-regulation of utrophin, alpha7B integrin, and dystroglycans. Ectopic ADAM12 expression in normal mouse skeletal muscle also improved regeneration after freeze injury, presumably by the same mechanism. Hence, it was suggested that ADAM12 could be a candidate for nonreplacement gene therapy of Duchenne muscular dystrophy. We therefore evaluated the long-term effect of ADAM12 overexpression in muscle. Surprisingly, we observed loss of skeletal muscle and accelerated fibrosis and adipogenesis in 1-year-old mdx mice transgenically overexpressing ADAM12 (ADAM12(+)/mdx mice), even though their utrophin levels were mildly elevated compared with age-matched controls. Thus, membrane stabilization was not sufficient to provide protection during prolonged disease. Consequently, we reinvestigated skeletal muscle regeneration in ADAM12 transgenic mice (ADAM12(+)) after a knife cut lesion and observed that the regeneration process was significantly impaired. ADAM12 seemed to inhibit the satellite cell response and delay myoblast differentiation. These results discourage long-term therapeutic use of ADAM12. They also point to impaired regeneration as a possible factor in development of muscular dystrophy.

Dissecting the signaling and mechanical functions of the dystrophin-glycoprotein complex

Duchenne muscular dystrophy is a severe disorder caused by mutations in the dystrophin gene. Dystrophin is required for assembly of the dystrophin-glycoprotein complex and provides a mechanically strong link between the cytoskeleton and the extracellular matrix. Several proteins in the complex also participate in signaling cascades, but the relationship between these signaling and mechanical functions in the development of muscular dystrophy is unclear. To explore the mechanisms of myofiber necrosis in dystrophin-deficient muscle, we tested the hypothesis that restoration of this complex without a link to the cytoskeleton ameliorates dystrophic pathology. Transgenic mice were generated that express Dp116, a non-muscle isoform of dystrophin that assembles the dystrophin-glycoprotein complex, in muscles of dystrophin-deficient mdx(4cv) mice. However, the phenotype of these mice was more severe than in controls. Displacement of utrophin by Dp116 correlated with the severity of dystrophy in different muscle groups. Comparison with other transgenic lines demonstrated that parts of the dystrophin central rod domain were required to localize neuronal nitric oxide synthase to the sarcolemma, but this was not correlated with presence or extent of dystrophy. Our results suggest that mechanical destabilization, rather than signaling dysfunction, is the primary cause of myofiber necrosis in dystrophin-deficient muscle.

Inhibition of dystroglycan binding to laminin disrupts the PI3K/AKT pathway and survival signaling in muscle cells.

Dystroglycan is a component of the dystrophin-glycoprotein complex (DGC) in muscle and a cell surface receptor for laminin. Numerous muscular dystrophies are the result of disruption of proteins comprising the DGC, but the underlying pathogenetic mechanisms are unknown. Because apoptosis is an early feature of muscular dystrophy in vivo, and perturbation of cell-extracellular matrix associations is known to induce apoptosis, we investigated the role of dystroglycan-laminin interactions in the propagation and maintenance of cell survival signals in muscle cells. We found that disrupting the interaction between alpha-dystroglycan and the extracellular matrix protein laminin induces apoptosis in muscle cells. This increase in apoptosis is mediated in part by caspase activation and can be blocked by a caspase-3 inhibitor. We demonstrate a role for the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway in muscle cell-survival signaling using a pharmacological inhibitor of PI3K. Treatment with this inhibitor resulted in decreased phosphorylation of AKT and its downstream effector glycogen synthase kinase (GSK)-3beta and induced apoptosis in muscle cell cultures. Disruption of dystroglycan-laminin interactions resulted in decreased phosphorylation of AKT and GSK-3beta. Furthermore, activation of AKT prior to the disruption of dystroglycan-laminin protected the muscle cells from the induction of apoptosis. These results support a role for the PI3K/AKT pathway in the propagation of cell-survival signals mediated by the DGC and provide new insight into the molecular pathogenesis associated with the development of muscular dystrophies.

Studies on the lipid composition of the fragmented sarcoplasmic reticulum of normal and dystrophic chickens.

SummaryThe increased cholesterol content and altered phospholipid composition of the dystrophic FSR seems to support the proposition that membrane alterations may be instrumental in the development of muscular dystrophy [Hsu, Q.-S., and Kaldor, Proc Soc. Exp. Biol. Med. 131, 1398 (1969); Martonosi, A., Proc. Soc. Exp. Biol. Med. 127, 824 (1968)].

Effects of various selenium compounds on the development of muscular dystrophy and other vitamin E dyscrasias in the chick.

Selenite, selenate, selenodicysteine, selenocystine, and selenomethionine, when administered orally, provided comparable protection against the development of nutritional muscular dystrophy in chicks fed a vitamin E deficient fat-free diet. When unsaturated lipid was included in the tocopherol-deficient basal ration, many of the control chicks not receiving selenium developed exudative diathesis as well as muscular dystrophy. Supplementation with either selenoamino acids, selenite, or selenate completely prevented exudates and provided the same degree of protection against muscle degeneration as obtained with the fat-free diet. Unexpectedly, however, when lard was fed, supplementation with selenite or selenate (but not the selenoamino acids), while alleviating exudates and dystrophy, provoked the onset of encephalomalacia.

FACTORS AFFECTING THE DEVELOPMENT OF NUTRITIONAL MUSCULAR DYSTROPHY IN NORTHERN ONTARIO

Fall calves from beef cows fed dystrophy-producing hay were found susceptible to the development of muscular dystrophy at an early age when exposed to severe outwintering conditions. Histopathological examination revealed granular discoid and hyaline degeneration of skeletal muscles in all the dystrophic calves and in two of the animals, hyaline degeneration of the heart was observed. All the affected calves died before 14 days of age. No appreciable rise in serum glutamic oxalacetic transaminase values were found for dystrophic calves, evidently due to the extreme rapidity in which myopathy developed and subsequent death ensued.Spring calves from dams fed dystrophy-producing hay during the winter developed myopathy after their release to pasture. A markedly higher incidence of dystrophy occurred in calves which had been reared outside and allowed normal exercise in comparison to calves which had been confined indoors, suggesting that the pasture effect is not related to an increase in physical activity or alteration of climate.Electrocardiography was found to be a useful tool for diagnosis of the cardiac myopathy frequently observed in dystrophic calves. Electrocardiograms of affected calves showed widening of the QT interval, and non-isoelectric ST and whipped T-wave configurations.

Incorporation of methionine sulfur into tissue proteins of guinea pigs with vitamin E deficiency

Examination of methionine-S35 incorporation into the tissue proteins demonstrated that to prevent secondary metabolic disturbances, associated indirectly with E-avitaminosis, an increased amount of vitamin A (8,000 I. U. a week instead of 2,000 I. U.) should be included into the E vitamin devoid diet of guinea pigs. However, this did not eliminate distinct E avitaminosis phenomena, notably the development of muscular dystrophy. In this connection there was a rise in specific radioactivity of muscular proteins in the experimental animals. Evidently, this was largely due to changes in the labeled methionine penetration into the cells of the skeletal musculature during E-avitaminosis, since according to the relative specific radioactivity data there was no difference between the proteins of control and those of the animals with E-avitaminosis.

The Efficacy of Sulphur Amino Acids and Related Compounds for Protection Against the Development of Muscular Dystrophy in the Chick

Chicks fed purified rations low in tocopherol and the sulphur amino acids develop muscular dystrophy in the breast musculature which can be prevented by supplementing the basal diet with methionine, cysteine, cystine, or alpha tocopherol acetate (Machlin and Shalkop, 1956; Jenkins et al., 1962). The mechanism(s) by which the sulphur amino acids and vitamin E prevent muscular degeneration are not known. Our knowledge that methionine is irreversibly converted to cysteine and cystine in the tissues of the bird suggests that the biopotency of methionine may be correlated to its ability to form these amino acids. The experiment reported here was a preliminary investigation of the relation of sulphur amino acid structure to the protection provided against the development of muscular dystrophy in the chick.Day-old, White Plymouth Rock pullets were fed a tocopherol and cystine-deficient basal ration containing 20% casein and 1% l-arginine HC1 (Jenkins et al., 1962) for one …