Proteomic Profiling of Naturally Protected Extraocular Muscles From
Total Page：16
File Type：pdf, Size：1020Kb
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by MURAL - Maynooth University Research Archive Library Biochemical and Biophysical Research Communications 396 (2010) 1024–1029 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Proteomic profiling of naturally protected extraocular muscles from the dystrophin-deficient mdx mouse Caroline Lewis, Kay Ohlendieck * Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland article info abstract Article history: Duchenne muscular dystrophy is the most frequent neuromuscular disorder of childhood. Although this x- Received 29 April 2010 linked muscle disease is extremely progressive, not all subtypes of skeletal muscles are affected in the same Available online 22 May 2010 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 pro- Keywords: tected muscles we have performed a comparative proteomic study of the established mdx mouse model Extraocular muscle of x-linked muscular dystrophy. Fluorescence difference in-gel electrophoretic analysis of 9-week-old dys- MDX trophin-deficient versus age-matched normal extraocular muscle, using a pH 4–7 gel range, identified out of Muscular dystrophy 1088 recognized protein spots a moderate expression change in only seven protein species. Desmin, apoli- Muscle proteomics Utrophin poprotein A-I binding protein and perilipin-3 were found to be increased and gelsolin, gephyrin, transaldol- Heat shock protein ase, 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 Ca2+-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 extraoc- ular muscles, probably due to the substitution of dystrophin by its autosomal homolog. Utrophin appears to prevent the loss of dystrophin-associated proteins and Ca2+-handling elements in extraocular muscle tis- sue. Interestingly, the adaptive mechanisms that cause the sparing of extraocular fibers seem to be closely linked to an enhanced cellular stress response. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction tein complex links the extracellular matrix protein laminin and the subsarcolemmal actin membrane cytoskeleton in healthy Dystrophinopathies are characterized by a primary deficiency in muscle fibers, the reduced presence of the DG-subcomplex is be- the Dp427 isoform of the membrane cytoskeletal protein dystro- lieved to play a central role in the degeneration of dystrophic mus- phin [1] and secondary abnormalities in sarcolemmal stability, cles. However, not all subtypes of skeletal muscles are affected in ion homeostasis, cellular signaling, excitation–contraction cou- the same way in x-linked muscular dystrophy. For example, intrin- pling, and numerous metabolic pathways [2]. Severely progressive sic laryngeal and certain distal muscle fibers, as well as extraocular Duchenne muscular dystrophy (DMD) and the more benign Becker muscle (EOM), are protected from a severely degenerative pheno- muscular dystrophy are allelic muscle diseases and are classified type in dystrophic organisms [5–11]. Although the contractile according to changes in the expression level and/or size of dystro- efficiency of EOM is weakened in various muscle-related patholo- phin [3]. The loss in Dp427 triggers the drastic reduction in sarco- gies, such as myasthenia gravis, myasthenic syndromes, botulism, lemmal glycoproteins, such as sarcoglycans and dystroglycans and myotonia congenita [12–14], they are usually spared in DMD (DG) which in turn weakens the integrity of the plasmalemma patients [15–17]. and leads to an increased frequency of membrane rupturing in dys- Therefore, the characterization of these naturally protected trophic muscle tissues [4]. Since the Dp427-associated glycopro- muscles may lead to the identification of constitutive properties or adaptive mechanisms that cause the sparing of EOM in dystro- Abbreviations: aBC, aB-crystallin; CSQ, calsequestrin; cvHsp, cardiovascular phinopathies [17]. The histological, biochemical, and physiological heat shock protein; DIGE, difference in-gel electrophoresis; DG, dystroglycan; DMD, profile of EOM is markedly different from other skeletal muscles Duchenne muscular dystrophy; Dp427, dystrophin protein of 427 kDa; EOM, [18–21]. During myogenesis, the protein factors responsible for extraocular muscle; Hsp, heat shock protein; MS, mass spectrometry; SERCA, specifying pre-myogenic progenitor cells in EOM seem to be sarcoplasmic or endoplasmatic Ca2+-ATPase; Up395, utrophin protein of 395 kDa. distinct from those in the trunk [22]. EOM represent a unique * Corresponding author. Fax: +353 (1) 708 3845. E-mail address: [email protected] (K. Ohlendieck). muscle allotype that controls the movements of the eye. Usually 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.052 C. Lewis, K. Ohlendieck / Biochemical and Biophysical Research Communications 396 (2010) 1024–1029 1025 fast-twitching muscles are not resistant to fatigue. However, EOM dystrophin gene [25]. Mildly affected EOM and severely dystrophic is an exception to this general rule of skeletal muscle physiology. diaphragm muscle from male 9-week-old mdx mice and normal EOM is capable of fast contraction patterns, but is also extremely tissues from age-matched C57 mice were obtained from the Biore- fatigue-resistance [23]. The coordinated action of six EOMs, i.e. source Unit of NUI Maynooth [7]. Mice were kept under standard Musculus rectus superior, M. rectus inferior, M. rectus medial, M. conditions and all procedures were performed in accordance with rectus lateral, M. obliquus superior, and M. obliquus inferior, enables Irish guidelines on the use of animals for scientific experiments. the eyeball to move about its vertical, horizontal, and anteroposte- Animals were sacrificed by cervical dislocation and muscle tissues rior axis. The complex excitation–contraction–relaxation cycle of quickly removed and quick-frozen in liquid nitrogen [27]. EOM ranges from slow vergence and pursuit movements to rapid saccades. Hence, based on their unique physiological properties 2.3. Preparation of crude muscle extracts and unusual anatomical structure, many myologists consider EOM as a separate class of skeletal muscles [24]. For the biochemical analysis of crude EOM and diaphragm Since Dp427-deficient EOM does not appear to be affected in a extracts, three dystrophic and three age-matched normal muscle major way, we have carried out a mass spectrometry (MS)-based specimens were pulverized by grinding tissue pieces in liquid nitro- proteomic comparison of normal versus mdx preparations. The gen using a mortar and pestle. Ground muscle was then solubilized mdx mouse represents an established animal model of DMD, which in lysis buffer with the ratio of 100 mg wet weight to 1 ml lysis buffer exhibits severe muscle degeneration in the diaphragm, segmental (7 M urea, 2 M thiourea, 4% CHAPS, 2% IPG buffer pH4–7, 2% (w/v) necrosis in leg muscles, and a mild phenotype in EOM [25]. Proteo- DTT). The lysis buffer contained a freshly prepared protease inhibitor mic profiling of Dp427-deficient leg and diaphragm muscles has re- cocktail [27]. EOM protein samples for DIGE analysis were placed in vealed drastic changes in the expression levels of adenylate kinase, buffer containing only the above listed concentrations of urea, thio- the luminal Ca2+-binding protein calsequestrin (CSQ), the cytosolic urea, and CHAPS. Following gentle rocking for 30 min, suspensions Ca2+-binding protein regucalcin, and the cardiovascular heat shock were centrifuged at 4 °C for 20 min at 20,000g and the protein con- protein (cvHsp), as well as a large number of metabolic and contrac- centration determined [28]. tile muscle proteins, as recently reviewed by Lewis et al. [2]. Here, we have used fluorescence difference in-gel electrophoresis (DIGE), one 2.4. Fluorescence DIGE analysis of the most powerful comparable techniques available in modern biochemistry [26], to study potential changes in protein expression Optimized fluorescence labeling and electrophoretic separation levels in Dp427-deficient versus normal EOM preparations. The conditions, as recommended by Karp and Lilley [29], were em- identification of only moderate changes in a few select proteins ployed to determine potential differences in the expression pattern agrees with the notion of a naturally protected phenotype. Impor- of the accessible part of the soluble proteome from control versus tantly, immunoblotting showed an enhanced abundance of stress dystrophic EOM. Labeling was carried out with 200 pmol of Cy3 proteins in mdx EOM, as well as an elevated concentration of the fluor dye per 25 lg protein [26]. An internal pooled standard of autosomal dystrophin homolog utrophin of 395 kDa (Up395), a every sample was prepared with the Cy5 fluor. Labeled samples comparable density of b-DG and rescued levels of CSQ. Hence, natu- were vortexed and incubated for 30 min on ice in the dark. The rally protected mdx EOM appears to escape fiber degeneration due reaction was quenched