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Caveolin-3 is a muscle specific membrane protein which is important in the formation of caveolae (caveolin-containing lipid rafts) and which has been linked to membrane trafficking and signal transduction events. Mutations in caveolin-3 cause limb girdle muscular dystrophy type 1C (LGMD1C) and rippling muscle disease. Caveolin-3 is transiently associated with the t-tubular membrane during muscle development and it is widely reported to be localised exclusively at the sarcolemma in mature muscle fibres. Using microscopy, caveolin-3 was localized to the t-tubule in adult mouse soleus muscle fibres. Male Long-Evans hooded rats (6-8 months old) were sacrificed using a lethal overdose of fluothane in accordance with the La Trobe University Ethics Committee and the extensor digitalis longus (EDL) and soleus (SOL) muscles were excised. From these muscles, segments of fibres were collected with either their sarcolemma present (intact) or with their sarcolemma removed by mechanically-skinning the fibres (skinned). We found a high expression of caveolin-3 in skinned fibres, supporting an intracellular pool of caveolin-3, likely localised to the t-tubular membrane . A higher expression of caveolin-3 was seen in both skinned and intact fibres from the predominantly type I soleus muscle compared to the fibres from the predominantly type II EDL muscle (eg., for intact fibres there was 200 ± 25% caveolin-3 in SOL fibres relative to that in EDL fibres, p < 0.05, n = 12, unpaired, two-tailed t-test). The relative amount of caveolin-3 in skinned and intact segments was quantified by comparing skinned and intact segments from the same fibre. For EDL and SOL fibres the amount of caveolin-3 in a skinned segment was 70-80% of that in the intact segment. The amount of caveolin-3 present in EDL skinned fibres was also analysed alongside their corresponding sarcolemmal portions and when expressed as a percentage of the total caveolin-3 present, 29 ± 9% (n = 6) of the caveolin-3 was found in the sarcolemma. As expected, caveolin-3 was found to be tightly bound in skeletal muscle fibres with none of the protein being present in various physiologically-based wash solutions (including very low and physiologically high Ca2+ concentrations) following washes of skinned fibres for up to 60 min. However, 80% or more of the detectable caveolin-3 became diffusible following a 30 min treatment with 1% Triton X-100 solution. These findings show that there is a greater expression of caveolin-3 in slow twitch muscle fibres compared with fast-twitch muscle fibres and that a greater proportion of the caveolin-3 is found inside muscle fibres than in the sarcolemma.
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