University of Heidelberg (2006) Proc Physiol Soc 4, PC2
Poster Communications: Nicholas S. Freestone1, George Dickson2, Sue C. Brown3
1. Pharmacy, Kingston University, Kingston-upon-Thames, Surrey, United Kingdom. 2. Biochemistry, Royal Holloway-University of London, Egham, Surrey, United Kingdom. 3. Dubowitz Neuromuscular Centre, Imperial College, London, London, United Kingdom.
Dystrophin and its associated protein and glycoprotein complex is widely thought to confer integrity on the muscle fibre membrane during repeated cycles of contraction and relaxation. In the present study dystrophin-deficient mdx myotubes were subjected to repetitive electrical stimulation of variable frequencies (0.3–3 Hz) to test their ability to respond to this mechanical challenge. Calcium (Ca2+) handling associated with these contractions in the mdx myotubes was compared with that in control (B10) myotubes by use of the calcium-sensitive fluorescent dye Indo-1 AM (4 μM). Each myotube that responded to electrical stimulation showed no change in it’s individual Ca2+ handling ability when stimulated to contract at 0.5 Hz for an hour or more. However, relative to control myotubes (n = 53), contractile mdx myotubes (n = 74) exhibited a significant (p = 0.004) elevation in resting levels of intracellular Ca2+ (1.03 ± 0.17 ratio units in mdx compared with 0.90 ± 0.27 in B10; mean ± SEM, significance assessed by two sample t test), but a significant decrease in amplitude of the electrically induced Ca2+ transient (0.38 ± 0.14 ratio units in mdx compared with 0.52 ± 0.36 in B10 myotubes, p=<0.0001). However, these differences were not associated with any alteration in the distribution of the sarcoplasmic/endoplasmic reticulum ATPases or ryanodine receptors as assessed by immunostaining and imaging with a Leica TCS4D confocal laser scanning microscope. Overall these findings confirm that Ca2+ levels are elevated in dystrophin-deficient myotubes, but do not support the hypothesis that repetitive cycles of contraction and relaxation directly compromise sarcolemmal integrity and lead to aberrant Ca2+ dynamics. Analyses of induced Ca2+ transients over extended periods of rapid repetitive electrical stimulation revealed stable resting and peak Ca2+ concentrations in both mdx and control (B10) myotubes.
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