Subproteomics analysis of Ca+-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 Ca(2+) buffering capacity has been shown to exist in the dystrophic sarcoplasmic reticulum, surprisingly no changes in the abundance of the main luminal Ca(2+) 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 Ca(2+)-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 Ca(2+)-binding proteins of the luminal sarcoplasmic reticulum is drastically reduced. This included the main terminal cisternae constituent, calsequestrin, and the previously implicated Ca(2+)-shuttle element, sarcalumenin. In contrast, the 'Stains-All'-positive protein spot, representing the cytosolic Ca(2+)-binding component, calmodulin, was not changed in dystrophin-deficient fibres. The reduced 2D 'Stains-All' pattern of luminal Ca(2+)-binding proteins in mdx preparations supports the calcium hypothesis of muscular dystrophy. The previously described impaired Ca(2+) buffering capacity of the dystrophic sarcoplasmic reticulum is probably caused by a reduction in luminal Ca(2+)-binding proteins, including calsequestrin.
Doran
Philip
P
Department of Biology, National University of Ireland, Maynooth, County Kildare, Ireland.
Dowling
Paul
P
Lohan
James
J
McDonnell
Karen
K
Poetsch
Stephan
S
Ohlendieck
Kay
K
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002155:Calsequestrin / Q000378:metabolism*; D016189:Dystrophin / Q000235:genetics / Q000502:physiology*; D015180:Electrophoresis, Gel, Two-Dimensional; D015151:Immunoblotting; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D018482:Muscle, Skeletal / Q000378:metabolism*; D009137:Muscular Dystrophy, Animal / Q000378:metabolism*; D040901:Proteomics; D013347:Subcellular Fractions
D002147:Calmodulin; D002155:Calsequestrin; D016189:Dystrophin; D008565:Membrane Proteins; C070323:sarcalumenin; D002118:Calcium