Abstract
In skeletal muscle cells, plasma membrane depolarization causes a rapid calcium release from the sarcoplasmic reticulum through ryanodine receptors triggering contraction. In Duchenne muscular dystrophy (DMD), a lethal disease that is caused by the lack of the cytoskeletal protein dystrophin, the cytosolic calcium concentration is known to be increased, and this increase may lead to cell necrosis. Here, we used myotubes derived from control and mdx mice, the murine model of DMD, to study the calcium responses induced by nicotinic acetylcholine receptor stimulation. The photoprotein aequorin was expressed in the cytosol or targeted to the plasma membrane as a fusion protein with the synaptosome-associated protein SNAP-25, thus allowing calcium measurements in a restricted area localized just below the plasma membrane. The carbachol-induced calcium responses were 4.5 times bigger in dystrophic myotubes than in control myotubes. Moreover, in dystrophic myotubes the carbachol-mediated calcium responses measured in the subsarcolemmal area were at least 10 times bigger than in the bulk cytosol. The initial calcium responses were due to calcium influx into the cells followed by a fast refilling/release phase from the sarcoplasmic reticulum. In addition and unexpectedly, the inositol 1,4,5-trisphosphate receptor pathway was involved in these calcium signals only in the dystrophic myotubes. This surprising involvement of this calcium release channel in the excitation-contraction coupling could open new ways for understanding exercise-induced calcium increases and downstream muscle degeneration in mdx mice and, therefore, in DMD.
Highlights
Duchenne muscular dystrophy (DMD)1 is an X-linked disease that affects about 1 in 3500 males
We show that nicotinic receptor stimulation triggers nearplasma membrane Ca2ϩ increases that depend on IP3 receptor activation in myotubes derived from mdx mice only, showing a non-common pathway for excitation-contraction coupling
As the removal of intracellular Ca2ϩ is required for the complete reconstitution of aequorin [41], SNAP-25 aequorinexpressing cells were preincubated in Ca2ϩ-free physiological salt solution (PSS) containing 0.1 mM EGTA for 1 h before performing the experiment
Summary
Duchenne muscular dystrophy (DMD)1 is an X-linked disease that affects about 1 in 3500 males. In skeletal muscle cells and myotubes, it has been shown that activation of nicotinic receptors leads to plasma membrane depolarization, which triggers a voltage-gated Ca2ϩ channel opening. These results indicate that Ca2ϩ responses measured with SNAP-25 aequorin were due to Ca2ϩ increases in a restricted area under the plasma membrane, presumably where BAPTA-AM had very limited access.
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