Sztretye Mónika, Singlár Zoltán, Balogh Norbert, Kis Gréta, Szentesi Péter, Angyal Ágnes, Balatoni Ildikó, Csernoch László, Dienes Beatrix
Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Department of Anatomy, Histology and Embryology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.
Front Physiol. 2020 Dec 21;11:601090. doi: 10.3389/fphys.2020.601090. eCollection 2020.
In mice a naturally occurring 12-bp deletion in the myostatin gene is considered responsible for the compact phenotype (Mstn, Cmpt) labeled by a tremendous increase in body weight along with signs of muscle weakness, easier fatigability, decreased Orai1 expression and store operated calcium entry (SOCE). Here, on the one hand, Cmpt fibers were reconstructed with venus-Orai1 but this failed to restore SOCE. On the other hand, the endogenous Orai1 was silenced in fibers from wild type C57Bl6 mice which resulted in ∼70% of Orai1 being silenced in whole muscle homogenates as confirmed by Western blot, accompanied by an inhibitory effect on the voltage dependence of SR calcium release that manifested in a slight shift toward more positive potential values. This maneuver completely hampered SOCE. Our observations are consistent with the idea that Orai1 channels are present in distinct pools responsible for either a rapid refilling of the SR terminal cisternae connected to each voltage-activated calcium transient, or a slow SOCE associated with an overall depletion of calcium in the SR lumen. Furthermore, when Cmpt cells were loaded with the mitochondrial membrane potential sensitive dye TMRE, fiber segments with depolarized mitochondria were identified covering on average 26.5 ± 1.5% of the fiber area. These defective areas were located around the neuromuscular junction and displayed significantly smaller calcium transients. The ultrastructural analysis of the Cmpt fibers revealed changes in the mitochondrial morphology. In addition, the mitochondrial calcium uptake during repetitive stimulation was higher in the Cmpt fibers. Our results favor the idea that reduced function and/or expression of SOCE partners (in this study Orai1) and mitochondrial defects could play an important role in muscle weakness and degeneration associated with certain pathologies, perhaps including loss of function of the neuromuscular junction and aging.
在小鼠中，肌生成抑制素基因自然发生的12个碱基对缺失被认为是导致紧凑表型（Mstn，Cmpt）的原因，该表型的特征是体重大幅增加，伴有肌肉无力、易疲劳、Orai1表达降低以及储存性钙内流（SOCE）减少。一方面，用维纳斯-Orai1重建了Cmpt纤维，但这未能恢复SOCE。另一方面，在野生型C57Bl6小鼠的纤维中沉默了内源性Orai1，如蛋白质免疫印迹法所证实，这导致全肌肉匀浆中约70%的Orai1被沉默，同时对肌浆网钙释放的电压依赖性产生抑制作用，表现为向更正电位值的轻微偏移。这一操作完全阻碍了SOCE。我们的观察结果与以下观点一致，即Orai1通道存在于不同的池中，分别负责与每个电压激活的钙瞬变相关的肌浆网终池的快速再填充，或与肌浆网腔内钙的总体消耗相关的缓慢SOCE。此外，当用线粒体膜电位敏感染料TMRE加载Cmpt细胞时，发现线粒体去极化的纤维段平均覆盖纤维面积的26.5±1.5%。这些缺陷区域位于神经肌肉接头周围，并且显示出明显较小的钙瞬变。对Cmpt纤维的超微结构分析揭示了线粒体形态的变化。此外，重复刺激期间Cmpt纤维中的线粒体钙摄取更高。我们的结果支持这样的观点，即SOCE相关蛋白（在本研究中为Orai1）功能和/或表达的降低以及线粒体缺陷可能在与某些病理状况相关的肌肉无力和退化中起重要作用，这可能包括神经肌肉接头功能丧失和衰老。
