Proteomic profiling of naturally protected extraocular muscles from the dystrophin-deficient mdx mouse

Abstract

a b s t r a c tDuchenne muscular dystrophy is the most frequent neuromuscular disorder of childhood. Although this xlinked muscle disease is extremely progressive, not all subtypes of skeletal muscles are affected in the same way. While extremities and trunk muscles are drastically weakened, extraocular muscles are usually spared in Duchenne patients. In order to determine the global protein expression pattern in these naturally protected muscles we have performed a comparative proteomic study of the established mdx mouse model of x-linked muscular dystrophy. Fluorescence difference in-gel electrophoretic analysis of 9-week-old dystrophin-deficient versus age-matched normal extraocular muscle, using a pH 4-7 gel range, identified out of 1088 recognized protein spots a moderate expression change in only seven protein species. Desmin, apolipoprotein A-I binding protein and perilipin-3 were found to be increased and gelsolin, gephyrin, transaldolase, and acyl-CoA dehydrogenase were shown to be decreased in mdx extraocular muscles. Immunoblotting revealed a drastic up-regulation of utrophin, comparable levels of b-dystroglycan and key Ca 2+ -regulatory elements, and an elevated concentration of small stress proteins in mdx extraocular muscles. This suggests that despite the lack of dystrophin only a limited number of cellular systems are perturbed in mdx extraocular muscles, probably due to the substitution of dystrophin by its autosomal homolog. Utrophin appears to prevent the loss of dystrophin-associated proteins and Ca 2+ -handling elements in extraocular muscle tissue. Interestingly, the adaptive mechanisms that cause the sparing of extraocular fibers seem to be closely linked to an enhanced cellular stress response.

Proteomic analysis of muscle affected by motor neuron degeneration: The wobbler mouse model of amyotrophic lateral sclerosis

“…The 2D-GE separation of fluorescently labeled proteins was carried out by using a total of 100 mg protein per DIGE gel. Isoelectric focusing was performed using 24 cm strips in a IPGphor system from Amersham Biosciences/GE Healthcare (Little Chalfont, Buckinghamshire, UK) using running conditions as previously described [26]. Following firstdimension separation, gel strips were equilibrated for 20 min in equilibration buffer containing 100 mM dithiothreitol, followed by 10 min of equilibration in buffer containing 250 mM ideoacetamide.…”

“…The voltage was set to 1700 V. Database searches were carried out with Mascot MS/MS Ion search (Matrix Science, London, UK). For data verification, immunoblotting was carried out as previously described in detail [26].…”

Understanding the molecular consequences of inherited muscular dystrophies: advancements through proteomic experimentation

“…One possible explanation for this apparent structural reorganisation is that it may represent an attempted compensatory response to stabilize the weakened cytoskeleton (54). It is interesting to note that increased levels of desmin were also detected in a proteomics study of the mildly-affected (and thus, non-regenerating) extraocular muscle from the mdx mouse (55), lending support to this notion. The possibility, however, that the changes in structural proteins may merely depict the ongoing process of cellular degeneration and / or fibrosis must also be considered (56).…”

“…A 2D-DIGE based quantitative proteomics comparison of the mildly affected EOM from mdx and control mice revealed differential expression of just seven proteins (55). The authors highlight how these results are a stark contrast to previous 2D-gel based comparisons of the severely affected diaphragm muscle, where between 20 and 35 differentially expressed proteins were detected (72,73).…”

“…The authors highlight how these results are a stark contrast to previous 2D-gel based comparisons of the severely affected diaphragm muscle, where between 20 and 35 differentially expressed proteins were detected (72,73). This suggests that there is a minimal perturbation of molecular pathways in the EOM muscle, and perhaps also implies that adaptive molecular pathways may not extend far beyond a straightforward upregulation of the dystrophin homologue, utrophin (55). Temporal proteomic studies of DMD: Several proteomic studies aimed to identify temporal changes during disease progression in DMD mouse models.…”