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U. Zeiger, M. H. J. Wiesen, C. H. Mitchell, T. S. Khurana; Extraocular Muscles Buffer Calcium Better Than Limb Muscle: Implications for Preferential Sparing in Duchenne's Muscular Dystrophy. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5277.
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Duchenne's Muscular Dystrophy (DMD) is the most common fatal, genetic disease in males and associated with widespread muscle wasting. Extraocular muscles (EOMs) are spared in DMD, although the reasons for this preferential sparing remain elusive. Dysregulation of calcium homeostasis has been suggested to contribute to muscle damage in DMD. Based on morphological observations, pharmacological tests and expression profiling, we propose that EOM sparing is facilitated by differential calcium buffering properties of EOM compared to limb (tibialis anterior-TA) muscle. We investigated the role of candidate proteins as well as compared calcium handling properties of cultured myotubes from EOM and TA.
The sarcoplasmic reticulum (SR) content of EOM and TA muscle was calculated from electron microscopy (EM) micrographs. Quantitative PCR was used to compare mRNA expression levels. Primary myoblasts from EOM and TA from 5-10-day old Sprague Dawley rats were cultured and fused in vitro, using the Rando & Blau (1994) protocol. Intracellular calcium buffering was determined using Fura-2 loaded myotubes after challenging the cells with ionomycin and 300nM extracellular Ca2+.
EM morphometry showed a 1.7 fold larger SR area in pale global EOM fibers than TA. The expression of mRNAs for several calcium and SR related proteins including SERCA1, Phospholamban and S100a1 was higher in EOM than TA. Cultured EOM myotubes showed 2.3 fold larger calcium peaks in response to sudden increases in calcium. However, the signal decayed 1.7 fold faster in EOM than in TA, implying a more efficient buffering from excessive calcium levels in these muscles.
Larger SR and differential expression of calcium buffering proteins may contribute to the improved ability of EOM to handle elevated intracellular calcium levels. This in turn would be predicted to protect EOM from the calcium-mediated damage noted in DMD limb muscles.
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