Proteome analysis of the dystrophin-deficient MDX diaphragm reveals a drastic increase in the heat shock protein cvHSP

Abstract

Duchenne muscular dystrophy is the most commonly inherited neuromuscular disorder in humans. Although the primary genetic deficiency of dystrophin in X-linked muscular dystrophy is established, it is not well-known how pathophysiological events trigger the actual fibre degeneration. We have therefore performed a DIGE analysis of normal diaphragm muscle versus the severely affected x-linked muscular dystrophy (MDX) diaphragm, which represents an established animal model of dystrophinopathy. Out of 2398 detectable 2-D protein spots, 35 proteins showed a drastic differential expression pattern, with 21 proteins being decreased, including Fbxo11-protein, adenylate kinase, b-haemoglobin and dihydrolipoamide dehydrogenase, and 14 proteins being increased, including cvHSP, aldehyde reductase, desmin, vimentin, chaperonin, cardiac and muscle myosin heavy chain. This suggests that lack of sarcolemmal integrity triggers a generally perturbed protein expression pattern in dystrophin-deficient fibres. However, the most significant finding was the dramatic increase in the small heat shock protein cvHSP, which was confirmed by 2-D immunoblotting. Confocal fluorescence microscopy revealed elevated levels of cvHSP in MDX fibres. An immunoblotting survey of other key heat shock proteins showed a differential expression pattern in MDX diaphragm. Stress response appears to be an important cellular mechanism in dystrophic muscle and may be exploitable as a new approach to counteract muscle degeneration.

Mass spectrometric identification of dystrophin isoform Dp427 by on-membrane digestion of sarcolemma from skeletal muscle

“…Subsequently, 100 ll of extraction buffer (5% formic acid/acetonitrile, 1:2, v/v) was added to the trypsin-treated membranes and gently agitated for 15 min at 37°C. All supernatant fractions were placed into fresh plastic tubes, and peptides were dried down by vacuum centrifugation [9]. Peptides were reconstituted in LC running buffer (0.1% formic acid), centrifuged for 20 min in 22-lm cellulose spin filter tubes to remove any membrane particles, and then aliquoted into LC-MS vials.…”

“…The membrane cytoskeletal element consists of an N-terminal actin-binding domain, a central spectrin-like rod domain with proline-rich hinge regions, a cysteine-rich domain, and a C-terminal region containing membrane-binding sites [4]. Although biochemical and cell biological studies have clearly shown that the primary genetic deficiency of dystrophin results in the drastic reduction of the dystrophin-associated glycoprotein complex in X-linked muscular dystrophy [5], recent large-scale proteomic studies have surprisingly not identified dystrophin isoform Dp427 in gel electrophoretically separated muscle preparations [6][7][8][9]. Mass spectrometric approaches also failed to identify integral dystrophin-associated glycoproteins in the accessible total muscle proteome [9].…”

“…Although biochemical and cell biological studies have clearly shown that the primary genetic deficiency of dystrophin results in the drastic reduction of the dystrophin-associated glycoprotein complex in X-linked muscular dystrophy [5], recent large-scale proteomic studies have surprisingly not identified dystrophin isoform Dp427 in gel electrophoretically separated muscle preparations [6][7][8][9]. Mass spectrometric approaches also failed to identify integral dystrophin-associated glycoproteins in the accessible total muscle proteome [9]. Thus, gel electrophoresis-based proteomics has revealed changes in many novel signature molecules of muscular dystrophy [10], such as the enzyme adenylate kinase [6], the luminal Ca 2+ -binding protein calsequestrin [7], the cytosolic Ca 2+ -binding protein regucalcin [8], and the small stress protein cvHsp [9], but appears not to be suitable for detecting alterations in elements of the dystrophin-glycoprotein complex.…”

“…Mass spectrometric approaches also failed to identify integral dystrophin-associated glycoproteins in the accessible total muscle proteome [9]. Thus, gel electrophoresis-based proteomics has revealed changes in many novel signature molecules of muscular dystrophy [10], such as the enzyme adenylate kinase [6], the luminal Ca 2+ -binding protein calsequestrin [7], the cytosolic Ca 2+ -binding protein regucalcin [8], and the small stress protein cvHsp [9], but appears not to be suitable for detecting alterations in elements of the dystrophin-glycoprotein complex. This is probably related to the fact that conventional two-dimensional gel electrophoresis underrepresents many low-abundance and membrane-associated protein species [11][12][13].…”

Comprehensive proteome analysis of human skeletal muscle in cachexia and sarcopenia: a pilot study

“…Besides physical and age‐dependent proteome changes, genetic diseases also perturb the normal proteome in muscles 21. Doran and colleagues investigated the effects of the inheritable neuromuscular disorder Duchenne muscular dystrophy using 2D gel electrophoresis and identified 35 at least two‐fold differentially regulated proteins comparing a normal with a dystrophin‐deficient diaphragm 22. Overall, these studies show a remarkable increase in proteins identifiable from skeletal muscle tissue as LC–MS/MS instrumentation advances.…”