Abstract
Malignant hyperthermia (MH) is a life-threatening syndrome triggered by volatile anesthetics, in which uncontrolled elevation of myoplasmic Ca2+ leads to hypercontracture of skeletal muscle and elevation of body temperature. Our recent study showed that azumolene, an analog of dantrolene used to treat MH, inhibits a component of store-operated Ca2+ entry (SOCE) coupled to activation of the ryanodine receptor in skeletal muscle (JBC 281: 33477, 2006). Given our previous observation that overexpression of calsequestrin-1 (CSQ1) suppressed SOCE in skeletal muscle (JBC 278: 3286, 2003), here we tested the hypothesis that reduced CSQ1 expression would enhance an azumolene-sensitive SOCE in this tissue. A shRNA probe specific for CSQ1 (JBC 281: 15772, 2006) was introduced into flexor digitorum brevis (FDB) muscles of living mice using electroporation. Individual transfected FDB muscle fibers labeled with a fluorescent marker were isolated for SOCE measurements using Mn-quenching of Fura-2 fluorescence. At room temperature (20–22°C), SOCE induced by caffeine/ryanodine was significantly enhanced in CSQ1-knockdown muscle fibers (in 10−4 ΔF360/s, 9.36 ± 1.31) compared to those transfected with control (4.71 ± 1.29, p<0.05). Pre-incubation with azumolene (20 μM) completely inhibited the elevated SOCE detected in CSQ1-knockdown fibers (1.26 ± 0.38, p<0.01). To prevent muscle contraction, we used N-benzyl-p-toluene sulfonamide (BTS, 40 μM), a specific myosin II inhibitor. When temperature of the bathing solution was increased to 40°C, muscle fibers with knockdown of CSQ1 displayed a significant elevation in cytosolic Ca2+ over that seen in control fibers. Thus reduced CSQ1 expression is likely coupled to elevation of cytosolic Ca2+ due to increased SOCE function at higher temperatures. These results suggest that elevated SOCE activity in skeletal muscle may be linked to the pathophysiology of MH and the heat-sensitivity of MH-susceptible animals.
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