Abstract
In mice a naturally occurring 12-bp deletion in the myostatin gene is considered responsible for the compact phenotype (MstnCmpt–dl1Abc, 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.
Highlights
Force generation, the main role of skeletal muscle is the result of muscle contraction and relaxation regulated by changes in the intracellular free Ca2+ level
In our earlier work (Sztretye et al, 2017) we have shown that endogenous Orai1 levels were reduced in the Cmpt flexor digitorum brevis (FDB) muscles which had direct functional consequences on store operated Ca2+ entry (SOCE) activity
The role of SOCE in maintaining contractile function of skeletal muscle during sustained stimulation has been widely studied and its contribution to SR refilling required for maintained calcium release during prolonged activity, and to fatigue resistance is generally accepted (Allen et al, 2008; Wei-Lapierre et al, 2013; Pan et al, 2014; Boncompagni et al, 2017)
Summary
The main role of skeletal muscle is the result of muscle contraction and relaxation regulated by changes in the intracellular free Ca2+ level. SOCE is coordinated by two key proteins: stromal interaction molecule 1 (STIM1) and Orai1 The former is the luminal Ca2+ level sensor in the SR membrane, while the latter is the Ca2+-release activated calcium (CRAC) channel located in the surface membrane (Desai et al, 2015; Vaeth et al, 2017). An increasing number of studies seem to contradict this idea presenting evidences on the role of SOCE in skeletal muscle contraction, development, fatigue reduction, replenishing SR stores during prolonged muscle contraction (Wei-Lapierre et al, 2013; Carrell et al, 2016; Boncompagni et al, 2017), in maintaining the SR calcium content during EC coupling (Sztretye et al, 2017; Koenig et al, 2018) even under conditions of sustained Ca2+ concentration in the SR (Koenig et al, 2019) and disease states (Stiber and Rosenberg, 2011)
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