Calcium-binding proteins in skeletal muscles of the mdx mice: potential role in the pathogenesis of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy is one of the most common hereditary diseases. Abnormal ion handling renders dystrophic muscle fibers more susceptible to necrosis and a rise in intracellular calcium is an important initiating event in dystrophic muscle pathogenesis. In the mdx mice, muscles are affected with different intensities and some muscles are spared. We investigated the levels of the calcium-binding proteins calsequestrin and calmodulin in the non-spared axial (sternomastoid and diaphragm), limb (tibialis anterior and soleus), cardiac and in the spared extraocular muscles (EOM) of control and mdx mice. Immunoblotting analysis showed a significant increase of the proteins in the spared mdx EOM and a significant decrease in the most affected diaphragm. Both proteins were comparable to the cardiac muscle controls. In limb and sternomastoid muscles, calmodulin and calsequestrin were affected differently. These results suggest that differential levels of the calcium-handling proteins may be involved in the pathogenesis of myonecrosis in mdx muscles. Understanding the signaling mechanisms involving Ca(2+)-calmodulin activation and calsequestrin expression may be a valuable way to develop new therapeutic approaches to the dystrophinopaties.

Figure 1

Histological appearance of control tibialis anterior (ctrl TA) and dystrophic (mdx) extraocular muscle (EOM) rectus, diaphragm (DIA) and heart. Control TA shows fibers with uniform diameter and peripheral nuclei. Dystrophic DIA shows areas of inflammatory reaction (asterisks) and fibers with central nuclei (arrows), indicative of muscle degeneration-regeneration. EOM and heart from mdx mice show no signs of muscle degeneration-regeneration. Scale bar: 20 μm (mdx EOM); 50 μm (ctrl TA and mdx heart) and 90 μm (mdx DIA).

Figure 2

Dystrophin (DYS) and calmodulin (CaM) immunofluorescence in control (CTRL) and dystrophic (mdx) extraocular muscle (EOM) rectus and tibialis anterior (TA). Normal sarcolemmal labeling with dystrophin antibody is present in control EOM. No dystrophin labeling is seen in mdx EOM. CaM showed a cytoplasmic pattern of distribution in EOM and TA. Scale bar: 30 μm (EOM) and 75 μm (TA).

Figure 3

Immunofluorescence localization of calsequestrin (CSQ) in controls (CTRL) and dystrophic (mdx) extraocular muscle (EOM) rectus, tibialis anterior (TA) and soleus (SOL). CSQ showed a cytoplasmatic pattern of distribution in CTRL and mdx muscles. Scale bar: 30 μm (EOM); 65 μm (TA) and 85 μm (SOL).

Figure 4

Immunoblot analysis of calmodulin (CaM) and calsequestrin (CSQ) levels in crude extracts of extraocular muscles (EOM), diaphragm (DIA), sternomastoid (STN), heart, soleus (SOL) and tibialis anterior (TA) muscles from control (ct) and dystrophic (mdx) mice. (a) Western blots of CaM and CSQ. (b) Same blot as (a) reprobed for GAPDH as a loading control. (c) Levels of calcium-binding proteins expressed in percentage normalized to the levels of GAPDH. The asterisk (*) means significantly different from control at P< 0.05. Error bars, SD.