Skeletal Muscle Of Dystrophic Mice Research Articles

Assessment of Muscle Function Following hiPSC-Derived Myoblast Transplantation in Dystrophic Mice.

Muscular dystrophies are caused by genetic variants in genes encoding for proteins important for muscle structure or function, leading to a loss of muscle integrity and muscle wasting. To this day, no cure has been found for these diseases. Different therapeutic approaches are under intensive investigation. Cellular therapy has been extensively studied for diseases such as Duchenne Muscular Dystrophy, a debilitating disease caused by a mutation in the DMD gene, encoding for the dystrophin protein. Healthy myogenic cells transplanted into dystrophic muscles have the potential to engraft at long-term and fuse to donate their nuclei to the dystrophin-deficient myofibers, thereby restoring normal gene expression. Despite promising preclinical studies, the clinical trials had limited success so far due to many technical limitations. The recent technological advances in induced-pluripotent stem cells and genome editing opened new opportunities in this field. One of the keys to efficiently translate these new technologies into clinical benefits is to use relevant endpoints for preclinical studies. Considering that dystrophic muscles are susceptible to contraction-induced injury, the assessment of their resistance to repeated eccentric contractions is an optimal outcome to evaluate their functional recovery following cell transplantation. This protocol describes the procedure to generate induced-pluripotent stem cell-derived myoblasts, transplant these cells into skeletal muscle of immunosuppressed dystrophic mice, and assess muscle function in situ by measuring the resistance of the transplanted muscle to repeated eccentric contractions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Generation of hiPSC-derived myoblasts. Basic Protocol 2: Transplantation of hiPSC-derived myoblasts in skeletal muscle of dystrophic mice. Basic Protocol 3: Assessment of muscle function in situ.

DMD TREATMENT: ANIMAL MODELS: P.205Effect of PDE5 inhibition on the post-contractile MRI blood-oxygenation-level-dependent (BOLD) effect in skeletal muscle of dystrophic mice

DMD TREATMENT: ANIMAL MODELS: P.205Effect of PDE5 inhibition on the post-contractile MRI blood-oxygenation-level-dependent (BOLD) effect in skeletal muscle of dystrophic mice

Altered bone-regulating myokine expression in skeletal muscle Of Duchenne muscular dystrophy mouse models.

Duchenne muscular dystrophy (DMD) has been well characterized as a disease that affects both skeletal muscle and bone. The pathophysiology responsible for the deficits in bone tissue is still unclear. Quantitative reverse-transcription polymerase chain reaction and Western blot analyses of known myokines from skeletal muscle were performed on dystrophic mouse models and wild-type (WT) controls to identify differentially expressed bone-regulating myokines. Twenty-four of 43 myokine genes demonstrated significantly different mRNA expression in the skeletal muscles of dystrophic mice when compared with muscles of WT mice. Several differently expressed bone-regulating myokine genes were identified, and their protein levels were also verified by Western blot. Dystrophic skeletal muscle demonstrated a significantly altered myokine gene expression profile. mRNA and protein levels of several bone-regulating myokines were significantly altered in dystrophic skeletal muscle, which suggests pathological role of bone-regulating myokines on bone homeostasis in DMD. Muscle Nerve 58: 573-582, 2018.

515. Mesenchymal Progenitor Cells (MPCs) Responsible for Fat Accumulation in Dystrophic Muscle May Also Impair the Myofiber Regeneration of Muscle Progenitors in Skeletal Muscle of Dystrophic Mice

515. Mesenchymal Progenitor Cells (MPCs) Responsible for Fat Accumulation in Dystrophic Muscle May Also Impair the Myofiber Regeneration of Muscle Progenitors in Skeletal Muscle of Dystrophic Mice

Disease-Related Modification of Chloride Conductance in Skeletal Muscle of Dystrophic Mice: Expression of CLC-1 Channel and Role of Inflammation and Oxidative Stress-Related Signaling

A decrease in resting chloride channel conductance (gCl) occurs in dystrophin-deficient myofibers of mdx mouse as a consequence of both spontaneous degeneration, as in diaphragm (DIA), or exercise-induced damage as in fast-twitch EDL muscle (De Luca et al., J. Pharmacol. Exp. Ther. 2003). Alterations in gCl, in parallel with markers of inflammation, can be contrasted by small restoration in dystrophin expression (De Luca et al., Neurobiol Dis, 2008). We focused on the molecular mechanisms underlying gCl impairment and its relation to primary defect. Preliminary qRT-PCR experiments showed a 30-35% reduction of CLC-1 mRNA in both DIA and EDL muscles of mdx mice, irrespective to exercise regimen. The reduction was consistent with the impairment of gCl detected in concomitant electrophysiological experiments in DIA and EDL myofibers; however the selective alteration of gCl in exercised mdx EDL muscle remained unexplained. Further experiments are ongoing to evaluate the possible outcome of pathology and mechanical stress on CLC-1 channel protein level and on the expression of other chloride channel types. Recent results showed that in vivo inhibition of angiotensin (Ang)-II contrasts the decrease in gCl in mdx EDL muscle, disclosing a possible role of this pro-inflammatory and pro-oxidative mediator in chloride channel function (Cozzoli et al., Pharmacol Res 2011). The application of Ang-II to wt EDL fibers reduced gCl in a concentration-dependent manner, with a half-maximal concentration of 67nM. The effect was inhibited by the AT1-receptor antagonist losartan, as well as by the PKC-inhibitor chelerythrine, the antioxidant N-acetyl-cysteine and the inhibitor of NADPH-oxidase apocynin. The results demonstrate that CLC-1 channel expression is affected in dystrophinopathies and that further modulation of gCl is related to inflammation and oxidative stress in skeletal muscle.

Quantification of Ceroid and Lipofuscin in Skeletal Muscle

Ceroid and lipofuscin are autofluorescent granules thought to be generated as a consequence of chronic oxidative stress. Because ceroid and lipofuscin are persistent in tissue, their measurement can provide a lifetime history of exposure to chronic oxidative stress. Although ceroid and lipofuscin can be measured by quantification of autofluorescent granules, current methods rely on subjective assessment. Furthermore, there has not been any evaluation of variables affecting quantitative measurements. The article describes a simple statistical approach that can be readily applied to quantitate ceroid and lipofuscin. Furthermore, it is shown that several factors, including magnification tissue thickness and tissue level, can affect precision and sensitivity. After optimizing for these factors, the authors show that ceroid and lipofuscin can be measured reproducibly in the skeletal muscle of dystrophic mice (ceroid) and aged mice (lipofuscin).

Use of imaging biomarkers to assess perfusion and glucose metabolism in the skeletal muscle of dystrophic mice

BackgroundDuchenne muscular dystrophy (DMD) is a severe neuromuscular disease that affects 1 in 3500 boys. The disease is characterized by progressive muscle degeneration that results from mutations in or loss of the cytoskeletal protein, dystrophin, from the glycoprotein membrane complex, thus increasing the susceptibility of contractile muscle to injury. To date, disease progression is typically assessed using invasive techniques such as muscle biopsies, and while there are recent reports of the use of magnetic resonance, ultrasound and optical imaging technologies to address the issue of disease progression and monitoring therapeutic intervention in dystrophic mice, our study aims to validate the use of imaging biomarkers (muscle perfusion and metabolism) in a longitudinal assessment of skeletal muscle degeneration/regeneration in two murine models of muscular dystrophy.MethodsWild-type (w.t.) and dystrophic mice (weakly-affected mdx mice that are characterized by a point mutation in dystrophin; severely-affected mdx:utrn-/- (udx) mice that lack functional dystrophin and are null for utrophin) were exercised three times a week for 30 minutes. To follow the progression of DMD, accumulation of 18 F-FDG, a measure of glucose metabolism, in both wild-type and affected mice was measured with a small animal PET scanner (GE eXplore Vista). To assess changes in blood flow and blood volume in the hind limb skeletal muscle, mice were injected intravenously with a CT contrast agent, and imaged with a small animal CT scanner (GE eXplore Ultra).ResultsIn hind limb skeletal muscle of both weakly-affected mdx mice and in severely-affected udx mice, we demonstrate an early, transient increase in both 18F-FDG uptake, and in blood flow and blood volume. Histological analysis of H&E-stained tissue collected from parallel littermates demonstrates the presence of both inflammatory infiltrate and centrally-located nuclei, a classic hallmark of myofibrillar regeneration. In both groups of affected mice, the early transient response was succeeded by a progressive decline in muscle perfusion and metabolism; this was also evidenced histologically.ConclusionsThe present study demonstrates the utility of non-invasive imaging biomarkers in characterizing muscle degeneration/regeneration in murine models of DMD. These techniques may now provide a promising alternative for assessing both disease progression and the efficacy of new therapeutic treatments in patients.

Lateral transmission of force is impaired in skeletal muscles of dystrophic mice and very old rats

Lateral transmission of force is impaired in skeletal muscles of dystrophic mice and very old rats

Lateral transmission of force is impaired in skeletal muscles of dystrophic mice and very old rats

The dystrophin–glycoprotein complex (DGC) provides an essential link from the muscle fibre cytoskeleton to the extracellular matrix. In dystrophic humans and mdx mice, mutations in the dystrophin gene disrupt the structure of the DGC causing severe damage to muscle fibres. In frog muscles, transmission of force laterally from an activated fibre to the muscle surface occurs without attenuation, but lateral transmission of force has not been demonstrated in mammalian muscles. A unique ‘yoke' apparatus was developed that attached to the epimysium of muscles midway between the tendons and enabled the measurement of lateral force. We now report that in muscles of young wild-type (WT) mice and rats, compared over a wide range of longitudinal forces, forces transmitted laterally showed little or no decrement. In contrast, for muscles of mdx mice and very old rats, forces transmitted laterally were impaired severely. Muscles of both mdx mice and very old rats showed major reductions in the expression of dystrophin. We conclude that during contractions, forces developed by skeletal muscles of young WT mice and rats are transmitted laterally from fibre to fibre through the DGC without decrement. In contrast, in muscles of dystrophic or very old animals, disruptions in DGC structure and function impair lateral transmission of force causing instability and increased susceptibility of fibres to contraction-induced injury.

Comparison of the subcellular localization of phosphodiesterase (PDE4) of normal and dystrophic mouse skeletal muscle

There are four genes that encode members of the cyclic nucleotide phosphodiesterase 4 (PDE4) family. Earlier work in the lab has shown that there is a decrease in particulate PDE4 activity between 5 and 15 weeks of age in skeletal muscle from the muscular dystrophy model mdx. Western blot bands in particulate fractions from mdx muscle show an age‐related decrease in PDE4 protein. In this study, PDE4 was examined immunohistochemically to determine whether the expression changes correspond to an alteration in subcellular localization. Skeletal muscle from hind legs was harvested and frozen in Neg‐50 tissue sectioning media, and sectioned in 10 µm thick longitudinal and cross‐sections. Sections were probed with an antiserum specific for PDE4A or an antiserum that recognizes multiple PDE4 families. Bound antibodies were visualized by confocal microscopy using a fluorescently‐tagged secondary antibody. The anti‐PDE4A antibody showed staining at the sarcolemma of 5 week‐old normal and mdx muscle. This staining was retained through 15 weeks. Imaging with the non‐selective PDE4 antiserum showed a stronger signal for PDE4 localized along the sarcolemma in 5 week‐old normal muscle compared to mdx muscle. These observations suggest that a PDE4 other than PDE4A is responsible for the observed age‐related differences in PDE4 expression seen in mdx muscle. This project was supported by NIH Grant 1R15AR050408‐01A2 from NIAMS.

Isolation and Characterization of Mesoangioblasts from Mouse, Dog, and Human Tissues

Mesoangioblasts are recently identified stem/progenitor cells, associated with small vessels of the mesoderm in mammals. Originally described in the mouse embryonic dorsal aorta, similar though not identical cells have been later identified and characterized from postnatal small vessels of skeletal muscle and heart (not described in this unit). They have in common the anatomical location, the expression of endothelial and/or pericyte markers, the ability to proliferate in culture, and the ability to undergo differentiation into various types of mesoderm cells upon proper culture conditions. Currently, the developmental origin of mesoangioblasts, their phenotypic heterogeneity, and the relationship with other mesoderm stem cells are not understood in detail and are the subject of active research. However, from a practical point of view, these cells have been successfully used in cell transplantation protocols that have yielded a significant rescue of structure and function in skeletal muscle of dystrophic mice and dogs. Since the corresponding human cells have been recently isolated and characterized, a clinical trial with these cells is planned in the near future. This unit provides detailed methods for isolation, culture, and characterization of mesoangioblasts.

Functional Coupling between TRPC3 and RyR1 Regulates the Expressions of Key Triadic Proteins

We have shown that TRPC3 (transient receptor potential channel canonical type 3) is sharply up-regulated during the early part of myotube differentiation and remains elevated in mature myotubes compared with myoblasts. To examine its functional roles in muscle, TRPC3 was "knocked down" in mouse primary skeletal myoblasts using retroviral-delivered small interference RNAs and single cell cloning. TRPC3 knockdown myoblasts (97.6 +/- 1.9% reduction in mRNA) were differentiated into myotubes (TRPC3 KD) and subjected to functional and biochemical assays. By measuring rates of Mn(2+) influx with Fura-2 and Ca(2+) transients with Fluo-4, we found that neither excitation-coupled Ca(2+) entry nor thapsigargin-induced store-operated Ca(2+) entry was significantly altered in TRPC3 KD, indicating that expression of TRPC3 is not required for engaging either Ca(2+) entry mechanism. In Ca(2+) imaging experiments, the gain of excitation-contraction coupling and the amplitude of the Ca(2+) release seen after direct RyR1 activation with caffeine was significantly reduced in TRPC3 KD. The decreased gain appears to be due to a decrease in RyR1 Ca(2+) release channel activity, because sarcoplasmic reticulum (SR) Ca(2+) content was not different between TRPC3 KD and wild-type myotubes. Immunoblot analysis demonstrated that TRPC1, calsequestrin, triadin, and junctophilin 1 were up-regulated (1.46 +/- 1.91-, 1.42 +/- 0.08-, 2.99 +/- 0.32-, and 1.91 +/- 0.26-fold, respectively) in TRPC3 KD. Based on these data, we conclude that expression of TRPC3 is tightly regulated during muscle cell differentiation and propose that functional interaction between TRPC3 and RyR1 may regulate the gain of SR Ca(2+) release independent of SR Ca(2+) load.

Decreased Mitochondrial Oxygen Consumption and Antioxidant Enzyme Activities in Skeletal Muscle of Dystrophic Mice after Low-Intensity Exercise

To evaluate low-intensity exercise training induced changes of mitochondrial metabolism in dystrophic skeletal muscle, oxygen consumption, reactive oxygen species (ROS) scavengers and antioxidant enzymes were measured in control (C57BL/10) and dystrophic (mdx) mice at 10 (young) and 22 (adult) weeks of age. Dystrophic and control mice were either kept sedentary or daily exercised on a treadmill (480 m/day, exercise training was initiated at 4 and 16 weeks of age for 6 weeks’ duration). Mitochondrial oxygen consumption was significantly lower in skeletal muscle from exercised young compared to sedentary young dystrophics. Whereas oxygen consumption was unchanged in exercised adult dystrophics, exercised adult controls exhibit a significant increase vs. sedentary adult controls. Contents of TBARS and lipofuscin were increased (+48%, +24%), while α-tocopherol concentration tended to decrease (p > 0.05) in exercised vs. sedentary young dystrophics. Compared to sedentary groups, glutathione peroxidase activity was decreased in exercised young dystrophic muscle (–12%), but increased in exercised controls (young controls +60%, adult controls +47%). In conclusion, adaptation to exercise-induced formation of ROS was limited in young dystrophic skeletal muscle but regained in that of adults.

IGF-I treatment improves the functional properties of fast- and slow-twitch skeletal muscles from dystrophic mice.

Although insulin-like growth factor-I (IGF-I) has been proposed for use by patients suffering from muscle wasting conditions, few studies have investigated the functional properties of dystrophic skeletal muscle following IGF-I treatment. 129P1 ReJ-Lama2(dy) (129 ReJ dy/dy) dystrophic mice suffer from a deficiency in the structural protein, laminin, and exhibit severe muscle wasting and weakness. We tested the hypothesis that 4 weeks of IGF-I treatment ( approximately 2 mg/kg body mass, 50 g/h via mini-osmotic pump, subcutaneously) would increase the mass and force producing capacity of skeletal muscles from dystrophic mice. IGF-I treatment increased the mass of the extensor digitorum longus (EDL) and soleus muscles of dystrophic mice by 20 and 29%, respectively, compared with untreated dystrophic mice (administered saline-vehicle only). Absolute maximum force (P(o)) of the EDL and soleus muscle was increased by 40 and 32%, respectively, following IGF-I treatment. Specific P(o) (sP(o)) was increased by 23% in the EDL muscles of treated compared with untreated mice, but in the soleus muscle sP(o) was unchanged. IGF-I treatment increased the proportion of type IIB and type IIA fibres and decreased the proportion of type I fibres in the EDL muscles of dystrophic mice. In the soleus muscles of dystrophic mice, IGF-I treatment increased the proportion of type IIA fibres and decreased the proportion of type I fibres. Average fibre cross-sectional area was increased in the EDL and soleus muscles of treated compared with untreated mice. We conclude that IGF-I treatment ameliorates muscle wasting and improves the functional properties of skeletal muscles of dystrophic mice. The findings have important implications for the role of IGF-I in ameliorating muscle wasting associated with the muscular dystrophies.

Antisense Inhibition of Decorin Expression in Myoblasts Decreases Cell Responsiveness to Transforming Growth Factor β and Accelerates Skeletal Muscle Differentiation

Decorin is a member of the family of the small leucine-rich proteoglycans. In addition to its function as an extracellular matrix organizer, it has the ability to activate the epidermal growth factor receptor, and it forms complexes with various isoforms of transforming growth factor beta (TGF-beta). Decorin is expressed during skeletal muscle differentiation and is up-regulated in dystrophic muscle. In this study we investigated the role of decorin in TGF-beta-dependent inhibition of myogenesis. To probe the function of decorin during myogenesis, C(2)C(12) myoblasts were stably transfected with a plasmid expressing antisense decorin mRNA. The resulting inhibition of decorin expression led to the expression of myogenin, a master transcription factor for muscle differentiation, under growth conditions and accelerated skeletal muscle differentiation as determined by the expression of creatine kinase. In contrast myogenin expression was inhibited by adenovirally induced decorin expression or by adding exogenous decorin. Reduced synthesis of decorin resulted in a 7-fold decreased sensitivity to TGF-beta-mediated inhibition of myogenin expression. In contrast, adenovirally induced decorin expression in wild type cells resulted in a 5-fold increased sensitivity to TGF-beta-mediated inhibition of myogenin expression. Transfection studies with the TGF-beta-dependent promoter of the plasminogen activator inhibitor-1 coupled with luciferase revealed that the transducing receptors for TGF-beta1 and TGF-beta2 were involved in the different responses of wild type and antisense decorin myoblasts. These results demonstrate that a reduction of decorin expression or of decorin availability results in a decreased responsiveness to TGF-beta. These findings strongly suggest a new role for decorin during skeletal muscle terminal differentiation by activating TGF-beta-dependent signaling pathways.

Synthesis of proteoglycans is augmented in dystrophic mdx mouse skeletal muscle

Mdx mice uniquely recover from degenerative dystrophic lesions through an intense myoproliferative response. The onset and progression of this process are controlled by a complex set of interactions between myoblasts and their environment. The presence of the extracellular matrix is essential for normal myogenesis. Proteoglycans are abundant components of the extracellular matrix. The synthesis of proteoglycans in mdx mice during skeletal muscle regeneration was evaluated. Incorporation of radioactive sulfate demonstrated a significant increase in the synthesis of several types of proteoglycans in mdx animals compared to age-matched controls. The size and charge of proteoglycans synthesized by the mdx mice remained unchanged. In particular, one of the up-regulated proteoglycans, the small chondroitin/dermatan sulfate proteoglycan decorin which is known to bind and to sequester transforming growth factor-beta, was investigated. Immunocytolocalization and in situ hybridization studies showed that decorin mainly accumulated in the endomysium, i.e. around individual skeletal muscle fibers from M. tibialis anterior and diaphragm.

C- myc is expressed in mouse skeletal muscle nuclei during post-natal maturation

Previous data have indicated the presence of c- myc mRNA and protein in mature skeletal muscle, but whether the protein is present as a Myc/Max heterodimer capable of influencing transcription in that tissue or its potential role in pathological muscle tissue has not been examined. The expression of c- myc in normal and mdx dystrophic mouse skeletal muscle was therefore investigated. C- myc mRNA was detected by Northern hybridisation in normal and dystrophic mouse muscle from mice up to 40 days of age and immunohistochemical staining confirmed that c- myc protein was expressed in muscle fibre nuclei in the muscles of mice up to 40 days of age. The presence of Myc-containing complexes that are able to bind to the concensus Myc/Max binding site was demonstrated in these muscles using the electrophoretic mobility shift assay. These results show that c- myc protein is expressed in functional complexes in both normal and dystrophic mouse skeletal muscle during post-natal maturation. They also show that expression of c- myc in mouse skeletal muscle decreases to undetectable levels by about 40 days of age. Although no differences were detected between the expression of c- myc in mdx and control mouse muscle, these data show that muscle contains Myc protein which has previously been demonstrated to be capable of initiating programmed cell death in other tissues.

Force and stiffness of old dystrophic (mdx) mouse skeletal muscles.

It has recently been suggested, based on studies of tissue pathology, that the limb muscles of old mdx mice may be a good model for the muscular changes seen in human Duchenne muscular dystrophy. To test this hypothesis, we measured force and stiffness of soleus and extensor digitorum longus (EDL) muscles of old (20-21 months) mdx mice and age-matched controls. The mdx and control muscles generated similar twitch, tetanic, and eccentric forces. They were also equally stiff. The results show that the mechanics of aged mdx limb muscles differ greatly from Duchenne muscular dystrophy in humans, and disagree with the hypothesis.

Purification and characterization of myonase from X-chromosome linked muscular dystrophic mouse skeletal muscle.

A chymotrypsin-like proteinase, designated myonase, was successfully purified to homogeneity from X-chromosome linked muscular dystrophic mouse skeletal muscle by affinity chromatography on agarose conjugated with lima bean trypsin inhibitor as ligand. The molecular mass of the purified myonase was determined to be 26 kDa by SDS-PAGE and to be 25,187 Da by mass spectrometry. The native enzyme is a single chain molecule and a monomeric protein without sugar side-chains. The nucleotide sequence of myonase mRNA is similar to mouse mast cell proteinase 4 (MMCP-4) cDNA. This is the first report of a native enzyme whose amino acid sequence closely corresponds to MMCP-4 cDNA. Myonase has chymotrypsin-like activities and hydrolyzes the amide bonds of synthetic substrates having Tyr and Phe residues at the P1 position. Myonase is most active at pH 9 and at high concentration of salts. Myonase preferentially hydrolyzes the Tyr4-Ile5 bond of angiotensin I and the Phe20-Ala21 bond of amyloid beta-protein, and it is less active towards the Phe8-His9 bond of angiotensin I and the Phe4-Ala5 and Tyr10-Glu11 bonds of amyloid beta-protein. Myonase is completely inhibited by such serine proteinase inhibitors as chymostatin, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but not by p-tosyl-L-phenylalanine chloromethyl ketone, p-tosyl-L-lysine chloromethyl ketone, pepstatin, E-64, EDTA, and o-phenanthroline. It is also inhibited by lima bean trypsin inhibitor, soy bean trypsin inhibitor, and human plasma alpha1-antichymotrysin. These properties match those of chymase, but unlike chymase, myonase does not interact with heparin in the regulation of its activity. Myonase was immunohistochemically localized in myocytes, but not in mast cells.

Presence of Laminin α5 Chain and Lack of Laminin α1 Chain during Human Muscle Development and in Muscular Dystrophies

There is currently a great interest in identifying laminin isoforms expressed in developing and regenerating skeletal muscle. Laminin alpha1 has been reported to localize to human fetal muscle and to be induced in muscular dystrophies based on immunohistochemistry with the monoclonal antibody 4C7, suggested to recognize the human laminin alpha1 chain. Nevertheless, there seems to be no expression of laminin alpha1 protein or mRNA in developing or dystrophic mouse skeletal muscle fibers. To address the discrepancy between the results obtained in developing and dystrophic human and mouse muscle we expressed the E3 domain of human laminin alpha1 chain as a recombinant protein and made antibodies specific for human laminin alpha1 chain (anti-hLN-alpha1G4/G5). We also made antibodies to the human laminin alpha5 chain purified from placenta. In the present report we show that hLN-alpha1G4/G5 antibodies react with a 400-kDa laminin alpha1 chain and that 4C7 reacts with a 380-kDa laminin alpha5 chain. Immunohistochemistry with the hLN-alpha1G4/G5 antibody and 4C7 revealed that the two antibodies stained human kidney, developing and dystrophic muscle in distinct patterns. Our data indicate that the previously reported expression patterns in developing, adult, and dystrophic human muscle tissues with 4C7 should be re-interpreted as an expression of laminin alpha5 chain. Our data are also consistent with earlier work in mouse, indicating that laminin alpha1 is largely an epithelial laminin chain not present in developing or dystrophic muscle fibers.