Drastic reduction of calsequestrin-like proteins and impaired calcium binding in dystrophicmdxmuscle

Abstract

Although the reduction in dystrophin-associated glycoproteins is the primary pathophysiological consequence of the deficiency in dystrophin, little is known about the secondary abnormalities leading to x-linked muscular dystrophy. As abnormal Ca2+ handling may be involved in myonecrosis, we investigated the fate of key Ca2+ regulatory membrane proteins in dystrophic mdx skeletal muscle membranes. Whereas the expression of the ryanodine receptor, the dihydropyridine receptor, the Ca2+-ATPase, and calsequestrin was not affected, a drastic decline in calsequestrin-like proteins of 150–220 kDa was observed in dystrophic microsomes using one-dimensional immunoblotting, two-dimensional immunoblotting with isoelectric focusing, diagonal two-dimensional blotting technique, and immunoprecipitation. In analogy, overall Ca2+ binding was reduced in the sarcoplasmic reticulum of dystrophic muscle. The reduction in Ca2+ binding proteins might be directly involved in triggering impaired Ca2+ sequestration within the lumen of the sarcoplasmic reticulum. Thus disturbed sarcolemmal Ca2+ fluxes seem to influence overall Ca2+homeostasis, resulting in distinct changes in the expression profile of a subset of Ca2+ handling proteins, which might be an important factor in the progressive functional decline of dystrophic muscle fibers.

Use of continuous-elution gel electrophoresis as a preparative tool for blot overlay analysis

“…These ion-binding elements are named calsequestrin-like proteins and are missing in muscular dystrophy (Fig. 6b), as has previously been reported [30]. The blot overlay analysis of dystrophic microsomes did not reveal a drastic difference in the binding pattern of peroxidase-conjugated calsequestrin.…”

Proteomic profiling of animal models mimicking skeletal muscle disorders

“…Skeletal muscles fibres from the mdx mouse have a point mutation in the dystrophin gene causing the near-to-total absence of the Dp427 isoform of this membrane cytoskeletal protein [90] and a concomitant reduction in dystrophin-associated glycoproteins such as dystroglycans (DGs), sarcoglycans (SG), syntrophins (SYN), sarcospan (SS) and dystrobrevins (DBs) [91]. In analogy to human dystrophic muscles, mdx fibres are characterized by varying degrees of necrosis, the appearance of regenerated myofibres with central nucleation, an increased susceptibility to os- motic shock, elevated levels of serum creatine kinase, stretch-induced injury, abnormal Ca 21 -handling and impaired (EC) coupling [91][92][93][94][95][96]. Figure 1 shows the histopathological analysis of dystrophin-deficient fibres.…”

“…However, the introduction of sarcolemmal Ca 21 -leak channels triggers a drastic elevation of cytosolic Ca 21 -levels [132], which in turn causes proteolysis and thereby muscle degeneration [131]. A complicating factor is the decreased Ca 21 -buffering capacity of the SR due to low levels of the Ca 21 -binding proteins calsequestrin (CSQ) [60], CSQ-like proteins [95] and sarcalumenin (SAR) [96], and impaired cystosolic Ca 21 -binding due to a reduction in regucalcin [41,61]. Since the cystosolic Ca 21 -concentration is the most important second messenger system with respect to many muscle functions including EC coupling, fibre relaxation, metabolic integration and fibre differentiation, abnormal Ca 21 -handling has severe pathological consequences [133].…”

Proteomic profiling of pathological and aged skeletal muscle fibres by peptide mass fingerprinting (Review)

“…In the case of dystrophinopathy, the natural MDX mutant mouse is widely employed as an animal model which exhibits severe muscular degeneration in the diaphragm (101). Muscle fibres from the MDX mouse exhibit, in analogy to human muscular dystrophy, a primary genetic abnormality in the dystrophin isoform Dp427 (102), a concomitant reduction in the dystrophin-associated glycoprotein complex (103), drastically elevated levels of serum creatine kinase (104), segmental fibre necrosis (105), an increased susceptibility to osmotic shock (106), stretchinduced injury (107) and an abnormal excitation-contractionrelaxation cycle (108)(109)(110). This makes MDX muscles not only a suitable system to study novel therapeutic strategies, but also to identify new biomarkers of X-linked muscular dystrophy.…”