Muscle degeneration without mechanical injury in sarcoglycan deficiency

In humans, mutations in the genes encoding components of the dystrophin-glycoprotein complex cause muscular dystrophy. Specifically, primary mutations in the genes encoding alpha-, beta-, gamma-, and delta-sarcoglycan have been identified in humans with limb-girdle muscular dystrophy. Mice lacking gamma-sarcoglycan develop progressive muscular dystrophy similar to human muscular dystrophy. Without gamma-sarcoglycan, beta- and delta-sarcoglycan are unstable at the muscle membrane and alpha-sarcoglycan is severely reduced. The expression and localization of dystrophin, dystroglycan, and laminin-alpha2, a mechanical link between the actin cytoskeleton and the extracellular matrix, appears unaffected by the loss of sarcoglycan. We assessed the functional integrity of this mechanical link and found that isolated muscles lacking gamma-sarcoglycan showed normal resistance to mechanical strain induced by eccentric muscle contraction. Sarcoglycan-deficient muscles also showed normal peak isometric and tetanic force generation. Furthermore, there was no evidence for contraction-induced injury in mice lacking gamma-sarcoglycan that were subjected to an extended, rigorous exercise regimen. These data demonstrate that mechanical weakness and contraction-induced muscle injury are not required for muscle degeneration and the dystrophic process. Thus, a nonmechanical mechanism, perhaps involving some unknown signaling function, likely is responsible for muscular dystrophy where sarcoglycan is deficient.     

How calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes

Duchenne muscular dystrophy patients lack the protein dystrophin which is an essential link in the complex of proteins that connect the cytoskeleton to the extracellular matrix. In mechanically stressed tissues such as muscle, transient sarcolemmal microdisruptions are normal, but in dystrophic muscle cells the frequency of these microdisruptions is greatly increased. Although both normal and dystrophic cells are able to actively repair these microdisruptions, calcium entry through the more frequent sarcolemmal microdisruptions of dystrophic cells results in an increased calcium-dependent proteolysis that alters the activity of the calcium leak channel. The accumulation of abnormally active calcium leak channels over time results in a gradual loss of calcium homeostasis and eventual cell death.     

Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model

Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorder, caused by recessive mutations in the dystrophin gene. One of every 3,500 males suffers from DMD, yet no treatment is currently available. Genetic therapeutic approaches, using primarily myoblast transplantation and adenovirus-mediated gene transfer, have met with limited success. Adeno-associated virus (AAV) vectors, although proven superior for muscle gene transfer, are too small (5 kb) to package the 14-kb dystrophin cDNA. Here we have created a series of minidystrophin genes (<4.2 kb) under the control of a muscle-specific promoter that readily package into AAV vectors. When injected into the muscle of mdx mice (a DMD model), two of the minigenes resulted in efficient and stable expression in a majority of the myofibers, restoring the missing dystrophin and dystrophin-associated protein complexes onto the plasma membrane. More importantly, this AAV treatment ameliorated dystrophic pathology in mdx muscle and led to normal myofiber morphology, histology, and cell membrane integrity. Thus, we have defined minimal functional dystrophin units and demonstrated the effectiveness of using AAV to deliver the minigenes in vivo, offering a promising avenue for DMD gene therapy.     

Convergent regulation of skeletal muscle Ca2+ channels by dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase

The skeletal muscle L-type Ca2+ channel (Ca(V)1.1), which is responsible for initiating muscle contraction, is regulated by phosphorylation by cAMP-dependent protein kinase (PKA) in a voltage-dependent manner that requires direct physical association between the channel and the kinase mediated through A-kinase anchoring proteins (AKAPs). The role of the actin cytoskeleton in channel regulation was investigated in skeletal myocytes cultured from wild-type mice, mdx mice that lack the cytoskeletal linkage protein dystrophin, and a skeletal muscle cell line, 129 CB3. Voltage dependence of channel activation was shifted positively, and potentiation was greatly diminished in mdx myocytes and in 129 CB3 cells treated with the microfilament stabilizer phalloidin. Voltage-dependent potentiation by strong depolarizing prepulses was reduced in mdx myocytes but could be restored by positively shifting the stimulus potentials to compensate for the positive shift in the voltage dependence of gating. Inclusion of PKA in the pipette caused a negative shift in the voltage dependence of activation and restored voltage-dependent potentiation in mdx myocytes. These results show that skeletal muscle Ca2+ channel activity and voltage-dependent potentiation are controlled by PKA and microfilaments in a convergent manner. Regulation of Ca2+ channel activity by hormones and neurotransmitters that use the PKA signal transduction pathway may interact in a critical way with the cytoskeleton and may be impaired by deletion of dystrophin, contributing to abnormal regulation of intracellular calcium concentrations in dystrophic muscle.     

Altered expression of dystrophin within the thoracic aorta in coarctation

Although persisting endothelial dysfunction has been established in the vasculature of patients following surgical repair of coarctation, it is unknown whether there are alterations in the cytoskeleton of the aorta in such patients. We compared staining of N-terminus dystrophin in the smooth muscle of the aortic wall of a patient with coarctation to that in a patient without coarctation, the latter undergoing surgical treatment of a double aortic arch. There was a marked difference in the pattern of expression of dystrophin between the two, with the coarcted specimen demonstrating marked fragmentation but normal intensity of staining. As far as we are aware, ours is the first report to demonstrate the presence of dystrophin in the smooth muscle of the aorta. Alterations in the cytoskeletal structure may account for underlying aberrations in endothelial function in such patients, and is a topic that warrants further investigation.     

The dystrophin glycoprotein complex: signaling strength and integrity for the sarcolemma

The dystrophin glycoprotein complex (DGC) is a specialization of cardiac and skeletal muscle membrane. This large multicomponent complex has both mechanical stabilizing and signaling roles in mediating interactions between the cytoskeleton, membrane, and extracellular matrix. Dystrophin, the protein product of the Duchenne and X-linked dilated cardiomyopathy locus, links cytoskeletal and membrane elements. Mutations in additional DGC genes, the sarcoglycans, also lead to cardiomyopathy and muscular dystrophy. Animal models of DGC mutants have shown that destabilization of the DGC leads to membrane fragility and loss of membrane integrity, resulting in degeneration of skeletal muscle and cardiomyocytes. Vascular reactivity is altered in response to primary degeneration in striated myocytes and arises from a vascular smooth muscle cell-extrinsic mechanism.     

Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways

AIMS: Cardiac myopathies are the second leading cause of death in patients with Duchenne and Becker muscular dystrophy, the two most common and severe forms of a disabling striated muscle disease. Although the genetic defect has been identified as mutations of the dystrophin gene, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Here, we examined cellular mechanisms linking the initial membrane damage to the dysfunction of dystrophic heart. METHODS AND RESULTS: Cardiac ventricular myocytes were enzymatically isolated from 5- to 9-month-old dystrophic mdx and wild-type (WT) mice. Cells were exposed to mechanical stress, applied as osmotic shock. Stress-induced cytosolic and mitochondrial Ca(2+) signals, production of reactive oxygen species (ROS), and mitochondrial membrane potential were monitored with confocal microscopy and fluorescent indicators. Pharmacological tools were used to scavenge ROS and to identify their possible sources. Osmotic shock triggered excessive cytosolic Ca(2+) signals, often lasting for several minutes, in 82% of mdx cells. In contrast, only 47% of the WT cardiomyocytes responded with transient and moderate intracellular Ca(2+) signals. On average, the reaction was 6-fold larger in mdx cells. Removal of extracellular Ca(2+) abolished these responses, implicating Ca(2+) influx as a trigger for abnormal Ca(2+) signalling. Our further experiments revealed that osmotic stress in mdx cells produced an increase in ROS production and mitochondrial Ca(2+) overload. The latter was followed by collapse of the mitochondrial membrane potential, an early sign of cell death. CONCLUSION: Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.     

Muscle involvement in juvenile idiopathic arthritis

OBJECTIVE: An observational study of changes in muscle structure and the relation to muscle strength in juvenile idiopathic arthritis (JIA). METHODS: Fifteen children and teenagers (eight girls and seven boys) with JIA, aged 9-19 yr (mean age 16.1), were studied. Muscle biopsies were obtained from the anterior tibial muscle and were examined using histopathological and immunohistochemical methods. Muscle fibre types were classified and fibre areas measured. As markers of inflammation, the major histocompatibility complex (MHC) class I and class II and the membrane attack complex (MAC) were analysed. Results were compared with biopsies from the gastrocnemius muscle in 33 young (19-23 yr) healthy controls. Isometric and isokinetic muscle strengths were measured in ankle dorsiflexion. Strength was compared with reference values for healthy age-matched controls. Nerve conduction velocities were recorded in the peroneal and sural nerves. RESULTS: Four of the 15 muscle biopsies were morphologically normal. Eleven biopsies showed minor unspecific changes. Two of these also showed minor signs of inflammation. MHC class II expression was found in 4/15 patients, which was significantly more than in the healthy controls (P = 0.0143). The expression of MHC class I and MAC did not differ from that in the controls. The mean area of type I fibres was lower than that of type IIA fibres in 12/13 biopsies. Muscle strength was significantly reduced in the patient group. There was a significant positive correlation between muscle fibre area and muscle strength. Nerve conduction studies were normal in all cases. CONCLUSIONS: Changes in leg muscle biopsies appear to be common in children and teenagers with JIA. The presence of inflammatory cells in the muscle and expression of MHC class II on muscle fibres may be a sign of inflammatory myopathy. There are no findings of type II muscle fibre hypotrophy or neuropathy, as in adults with RA.     

Dystrophin diagnosis: comparison of dystrophin abnormalities by immunofluorescence and immunoblot analyses.

Immunoblot characterization and immunofluorescence localization of dystrophin are presented for 76 human patients with various neuromuscular diseases. Normal dystrophin (shown by immunoblotting) was invariably visualized as a continuous, peripheral membrane immunostaining of myofibers. Biochemical abnormalities of dystrophin (either lower or higher molecular weight dystrophin) resulted in patchy, discontinuous immunostaining, suggesting that the abnormal dystrophin proteins are not capable of creating a complete membrane cytoskeleton network. There was a very strong correlation of clinical diagnoses with the type of dystrophin abnormality; all Duchenne muscular dystrophy patient muscle contained no detectable dystrophin, Becker muscular dystrophy patient muscle had clearly abnormal dystrophin, and unrelated diseases showed normal dystrophin. However, a single patient of five carrying the diagnosis of Fukuyama dystrophy showed no detectable dystrophin and thus appeared to be a Duchenne dystrophy patient by the biochemical assays. We know of no other case of a patient with a disease thought to be unrelated to Duchenne/Becker dystrophy yet demonstrating dystrophin deficiency. Based on the data presented, we conclude that immunofluorescence is the best technique for the detection of female carriers of Duchenne dystrophy, whereas immunoblotting appears superior for the prognostic diagnosis of Becker muscular dystrophy.     

Elevated subsarcolemmal Ca2+ in mdx mouse skeletal muscle fibers detected with Ca2+-activated K+ channels

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The cellular mechanisms responsible for the progressive skeletal muscle degeneration that characterizes the disease are still debated. One hypothesis suggests that the resting sarcolemmal permeability for Ca(2+) is increased in dystrophic muscle, leading to Ca(2+) accumulation in the cytosol and eventually to protein degradation. However, more recently, this hypothesis was challenged seriously by several groups that did not find any significant increase in the global intracellular Ca(2+) in muscle from mdx mice, an animal model of the human disease. In the present study, using plasma membrane Ca(2+)-activated K(+) channels as subsarcolemmal Ca(2+) probe, we tested the possibility of a Ca(2+) accumulation at the restricted subsarcolemmal level in mdx skeletal muscle fibers. Using the cell-attached configuration of the patch-clamp technique, we demonstrated that the voltage threshold for activation of high conductance Ca(2+)-activated K(+) channels is significantly lower in mdx than in control muscle, suggesting a higher subsarcolemmal [Ca(2+)]. In inside-out patches, we showed that this shift in the voltage threshold for high conductance Ca(2+)-activated K(+) channel activation could correspond to a approximately 3-fold increase in the subsarcolemmal Ca(2+) concentration in mdx muscle. These data favor the hypothesis according to which an increased calcium entry is associated with the absence of dystrophin in mdx skeletal muscle, leading to Ca(2+) overload at the subsarcolemmal level.     

Prevention of dystrophin-deficient cardiomyopathy in twenty-one-month-old carrier mice by mosaic dystrophin expression or complementary dystrophin/utrophin expression

A cure for dystrophin-deficient muscular dystrophy requires treating both skeletal muscle and the heart. Whereas mosaic dystrophin expression has been shown to protect skeletal muscle, controversy exists over whether mosaic expression is protective in the heart. We have shown recently that mosaic dystrophin expression prevents stress-induced heart damage in young carrier mice. Although an interesting finding, the clinical relevance remains to be established because young dystrophin-null mdx mice do not have heart disease. On the other hand, heart failure has been reported in human carriers. To resolve this mouse/human discrepancy, we evaluated the cardiac phenotype in 21-month-old mdx, carrier, and normal mice. We found dilated cardiomyopathy in old mdx mice but not in age-matched carrier mice. All anatomical parameters and physiological assay results (ECG and closed-chest Millar catheter) were within the normal range in old carrier mice. Focal myocardial inflammation was found in a small fraction of old carrier mice, but it had no major impact on heart function. Dobutamine stress revealed a near normal hemodynamic profile except for a marginal reduction in systolic pressure in old carrier mice. Immunostaining and Western blot showed dystrophin expression in 50% cardiomyocytes in old carrier mice. Interestingly, utrophin was upregulated in dystrophin-negative heart cells in carrier mice. In summary, we have provided the first clear-cut evidence that dilated cardiomyopathy in old mdx mice was prevented by mosaic dystrophin expression or complementary dystrophin/utrophin expression. Our results raise the hope for ameliorating dystrophic cardiomyopathy through partial gene and/or cell therapy.     

Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer

Antisense oligonucleotide-mediated exon skipping is able to correct out-of-frame mutations in Duchenne muscular dystrophy and restore truncated yet functional dystrophins. However, its application is limited by low potency and inefficiency in systemic delivery, especially failure to restore dystrophin in heart. Here, we conjugate a phosphorodiamidate morpholino oligomer with a designed cell-penetrating peptide (PPMO) targeting a mutated dystrophin exon. Systemic delivery of the novel PPMO restores dystrophin to almost normal levels in the cardiac and skeletal muscles in dystrophic mdx mouse. This leads to increase in muscle strength and prevents cardiac pump failure induced by dobutamine stress in vivo. Muscle pathology and function continue to improve during the 12-week course of biweekly treatment, with significant reduction in levels of serum creatine kinase. The high degree of potency of the oligomer in targeting all muscles and the lack of detectable toxicity and immune response support the feasibility of testing the novel oligomer in treating Duchenne muscular dystrophy patients.     

Isolated dystrophin molecules as seen by electron microscopy.

Dystrophin, the protein product of the Duchenne muscular dystrophy locus [Hoffman, E. P., Brown, R. H., Jr., & Kunkel, L. M. (1987) Cell 51, 919-928], is expressed in striated and smooth muscles as well as in non-muscle tissues. Examination of its primary structure has revealed that the molecule is composed of four domains, three of which share many features with the membrane cytoskeletal proteins spectrin and actinin. Dystrophin has thus been predicted to adopt a rod shape [Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228]. In the present study, we describe its isolation from the chicken gizzard smooth muscle and present electron microscopic images of the molecule. Polyclonal antibodies were first prepared from a dystrophin fragment derived from the chicken skeletal muscle gene (residues 1173-1728). A dystrophin-enriched membrane preparation from chicken gizzard muscle was then purified by passing it through an affinity chromatography column made with the anti-dystrophin antibodies. Electron microscopy of isolated and rotatory-shadowed dystrophin molecules revealed that the lengths measured for the dystrophin monomers (175 +/- 15 nm) are compatible with a structural arrangement of the repeat sequence segments in triple-barrel alpha-helices connected by short-turn regions, as was earlier postulated for the repeat domains of spectrin and actinin. Electron microscopic images indicate that in addition the dystrophin molecules could present the same capacity of self-association in oligomeric structures as these cytoskeletal proteins and may thus be a part of a complex molecular meshwork essential to muscle cell function.     

Human adhalin is alternatively spliced and the gene is located on chromosome 17q21.

Mutations in the dystrophin gene cause the X chromosome-linked, recessive Duchenne and Becker muscular dystrophies. Dystrophin, a large cytoskeletal protein, copurifies with a complex of dystrophin-associated proteins which serve to anchor dystrophin to the sarcolemma. One of these associated proteins, adhalin, has been implicated as a candidate for severe childhood autosomal recessive muscular dystrophy (SCARMD) due to absence of anti-adhalin staining in muscle biopsy samples taken from SCARMD patients. Furthermore, the Duchenne-like dystrophic phenotype seen in the SCARMD families was shown to be tightly linked to chromosome 13 markers. To determine the genetic mutation responsible for autosomal dystrophy, we characterized the human adhalin gene. Contrary to our expectation, human adhalin was mapped to chromosome 17q21, excluding adhalin as the gene causing chromosome 13-associated SCARMD. Additionally, a splice form of adhalin message was found that predicts a 35-kDa nontransmembrane adhalin. The expression of both adhalin splice forms is exclusively restricted to striated muscle, unlike other components of the dystrophin-glycoprotein complex.     

Functional muscle ischemia in neuronal nitric oxide synthase-deficient skeletal muscle of children with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutation of the gene encoding the cytoskeletal protein dystrophin. Despite a wealth of recent information about the molecular basis of DMD, effective treatment for this disease does not exist because the mechanism by which dystrophin deficiency produces the clinical phenotype is unknown. In both mouse and human skeletal muscle, dystrophin deficiency results in loss of neuronal nitric oxide synthase, which normally is localized to the sarcolemma as part of the dystrophin-glycoprotein complex. Recent studies in mice suggest that skeletal muscle-derived nitric oxide may play a key role in the regulation of blood flow within exercising skeletal muscle by blunting the vasoconstrictor response to alpha-adrenergic receptor activation. Here we report that this protective mechanism is defective in children with DMD, because the vasoconstrictor response (measured as a decrease in muscle oxygenation) to reflex sympathetic activation was not blunted during exercise of the dystrophic muscles. In contrast, this protective mechanism is intact in healthy children and those with polymyositis or limb-girdle muscular dystrophy, muscle diseases that do not result in loss of neuronal nitric oxide synthase. This clinical investigation suggests that unopposed sympathetic vasoconstriction in exercising human skeletal muscle may constitute a heretofore unappreciated vascular mechanism contributing to the pathogenesis of DMD.     

Fundamental Investigations of Bond Behaviour of High-Strength Micro Steel Fibres in Ultra-High Performance Concrete under Cyclic Tensile Loading

The objective of the contribution is to understand the fatigue bond behaviour of brass-coated high-strength micro steel fibres embedded in ultra-high performance concrete (UHPC). The study contains experimental pullout tests with variating parameters like load amplitude, fibre orientation, and fibre-embedded length. The test results show that fibres are generally pulled out of the concrete under monotonic loading and rupture partly under cyclic tensile loading. The maximum tensile stress per fibre is approximately 1176 N/mm², which is approximately one third of the fibre tensile strength (3576 N/mm²). The load-displacement curves under monotonic loading were transformed into a bond stress-slip relationship, which includes the effect of fibre orientation. The highest bond stress occurs for an orientation of 30° by approximately 10 N/mm². Under cyclic loading, no rupture occurs for fibres with an orientation of 90° within 100,000 load changes. Established S/N-curves of 30°- and 45°-inclined fibres do not show fatigue resistance of more than 1,000,000 load cycles for each tested load amplitude. For the simulation of fibre pullout tests with three-dimensional FEM, a model was developed that describes the local debonding between micro steel fibre and the UHPC-matrix and captures the elastic and inelastic stress-deformation behaviour of the interface using plasticity theory and a damage formulation. The model for the bond zone includes transverse pressure-independent composite mechanisms, such as adhesion and micro-interlocking and transverse pressure-induced static and sliding friction. This allows one to represent the interaction of the coupled structures with the bond zone. The progressive cracking in the contact zone and associated effects on the fibre load-bearing capacity are the decisive factors concerning the failure of the bond zone. With the developed model, it is possible to make detailed statements regarding the stress-deformation state along the fibre length. The fatigue process of the fibre-matrix bond with respect to cyclic loading is presented and analysed in the paper.