Fig. 5.

Calcium accumulation at motor endplates differs with transgenic expression of distinct AChR mutations.A, Sections of forelimb flexor muscles from resting δS262T, αC418W, αL251T, and εL269F mice demonstrate differences in degree of GBHA staining for different transgenic lines. Sections in the left panels were stained for cholinesterase to localize endplates (cholinesterase is brown) (Namba et al., 1967) and counterstained with hematoxylin and eosin. The adjacent serial sections in the right panels were stained for glyoxal bis 2 hydroxyanil (Evans, 1974) (GBHA, dark red or black) and methylene blue. There is no GBHA staining at control (data not shown) or δS262T endplates. Scale bar, 25 μm. B, Calcium accumulation at motor endplates differs according to mutation and increases with exercise. The proportions of motor endplates stained histochemically with GBHA in αC418W, αL251T, and εL269F mice at rest or after exercise is displayed in vertical barslabeled below. Dark bars indicate the lines that develop endplate myopathy (Fig. 6). At rest, no GBHA stain was detected at control or δS262T endplates, whereas 0.47% were labeled in αC418W mice, 2.8% in αL251T, and 19% in εL269F mice. The proportion of Ca²⁺-overloaded endplates detected in resting αL251T mice (n = 6) was slightly greater than that in αC418W mice (0.47%; p = 0.08;n = 6). The proportion of GBHA-stained endplates in resting εL269F transgenic mice was significantly greater than that of the other resting mice (p ≪ 0.001;n = 5). After exercise (see Materials and Methods), there was still no GBHA staining at motor endplates or elsewhere in muscle fibers of wild-type or δS262T transgenic mice, whereas marked increases in GBHA staining were seen in the other three transgenic lines after exercise. In exercised αC418W transgenic mice (n = 5), the proportion of endplates stained for GBHA increased to 3.9%, an eightfold increase (p < 0.01). In αL251T transgenic mice (n = 4) and εL269F transgenic mice (n = 4), the proportion of GBHA-stained endplates increased to 26% (10-fold; p < 0.002) and 33% (1.7-fold; p < 0.04), respectively. The proportions of Ca²⁺-overloaded endplates after exercise in αL251T transgenic mice and εL269F transgenic mice were significantly greater than the Ca²⁺-overloaded endplates in exercised αC418W transgenic mice (p < 0.01).