肢带肌营养不良2A(LGMD2A)是由编码骨骼肌特异性Calpain3的CAPN3基因突变引起的,Ca2+依赖性蛋白酶。钙蛋白酶3在三联体中的定位表明它有助于Ca2稳态。通过活细胞Ca2+测量,肌肉力学,免疫荧光,Capn3缺陷(C3KO)和野生型(WT)小鼠的电子显微镜(EM),我们确定钙蛋白酶3的丢失是否改变了储存操作的钙进入(SOCE)活性。直接的Ca2内流测量显示Capn3的损失导致静息SOCE升高和静息胞质Ca2增加,由EM观察到的钙进入单位(CEU)的高发生率支持。对C3KO和WT小鼠进行一次跑步机跑步以引起SOCE。在跑步机后运行的1HR内,在重复刺激期间,C3KO小鼠在指长伸肌中的力产生减少,而在指短屈肌肌纤维中的Ca2瞬变衰减更大。C3KO小鼠运动诱导的SOCE激活受损的惊人证据包括关键SOCE蛋白的共定位不良,基质相互作用分子1(STIM1)和ORAI1,并伴有C3KO肌肉中CEU的消失。这些结果表明,钙蛋白酶3是骨骼肌中SOCE的关键调节剂,并将SOCE失调鉴定为LGMD2A病理的促成因素。
Limb-Girdle Muscular Dystrophy 2A (LGMD2A) is caused by mutations in the CAPN3 gene encoding Calpain 3, a skeletal-muscle specific, Ca2+-dependent protease. Localization of Calpain 3 within the triad suggests it contributes to Ca2+ homeostasis. Through live-cell Ca2+ measurements, muscle mechanics, immunofluorescence, and electron microscopy (EM) in Capn3 deficient (C3KO) and wildtype (WT) mice, we determined if loss of Calpain 3 altered Store-Operated Calcium Entry (SOCE) activity. Direct Ca2+ influx measurements revealed loss of Capn3 elicits elevated resting SOCE and increased resting cytosolic Ca2+, supported by high incidence of calcium entry units (CEUs) observed by EM. C3KO and WT mice were subjected to a single bout of treadmill running to elicit SOCE. Within 1HR post-treadmill running, C3KO mice exhibited diminished force production in extensor digitorum longus muscles and a greater decay of Ca2+ transients in flexor digitorum brevis muscle fibers during repetitive stimulation. Striking evidence for impaired exercise-induced SOCE activation in C3KO mice included poor colocalization of key SOCE proteins, stromal-interacting molecule 1 (STIM1) and ORAI1, combined with disappearance of CEUs in C3KO muscles. These results demonstrate that Calpain 3 is a key regulator of SOCE in skeletal muscle and identify SOCE dysregulation as a contributing factor to LGMD2A pathology.