Subproteomics analysis of Ca2+-binding proteins demonstrates decreased calsequestrin expression in dystrophic mouse skeletal muscle

Duchenne muscular dystrophy represents one of the most common hereditary diseases. Abnormal ion handling is believed to render dystrophin-deficient muscle fibres more susceptible to necrosis. Although a reduced Ca2+ buffering capacity has been shown to exist in the dystrophic sarcoplasmic reticulum, surprisingly no changes in the abundance of the main luminal Ca2+ reservoir protein calsequestrin have been observed in microsomal preparations. To address this unexpected finding and eliminate potential technical artefacts of subcellular fractionation protocols, we employed a comparative subproteomics approach with total mouse skeletal muscle extracts. Immunoblotting, mass spectrometry and labelling of the entire muscle protein complement with the cationic carbocyanine dye 'Stains-All' was performed in order to evaluate the fate of major Ca2+-binding proteins in dystrophin-deficient skeletal muscle fibres. In contrast to a relatively comparable expression pattern of the main protein population in normal vs. dystrophic fibres, our analysis showed that the expression of key Ca2+-binding proteins of the luminal sarcoplasmic reticulum is drastically reduced. This included the main terminal cisternae constituent, calsequestrin, and the previously implicated Ca2+-shuttle element, sarcalumenin. In contrast, the 'Stains-All'-positive protein spot, representing the cytosolic Ca2+-binding component, calmodulin, was not changed in dystrophin-deficient fibres. The reduced 2D 'Stains-All' pattern of luminal Ca2+-binding proteins in mdx preparations supports the calcium hypothesis of muscular dystrophy. The previously described impaired Ca2+ buffering capacity of the dystrophic sarcoplasmic reticulum is probably caused by a reduction in luminal Ca2+-binding proteins, including calsequestrin.