Abstract
Stromal interaction molecule 1 (STIM1) mediates extracellular Ca2+ entry into the cytosol through a store-operated Ca2+ entry (SOCE) mechanism, which is involved in the physiological functions of various tissues, including skeletal muscle. STIM1 is also associated with skeletal muscle diseases, but its pathological mechanisms have not been well addressed. The present study focused on examining the pathological mechanism(s) of a mutant STIM1 (R429C) that causes human muscular hypotonia. R429C was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-cell Ca2+ imaging of myotubes and transmission electron microscopy (TEM) along with biochemical approaches. R429C did not interfere with the terminal differentiation of myoblasts to myotubes. Unlike wild-type STIM1, there was no further increase of SOCE by R429C. R429C bound to endogenous STIM1 and slowed down the initial rate of SOCE that were mediated by endogenous STIM1. Moreover, R429C increased intracellular Ca2+ movement in response to membrane depolarization by eliminating the attenuation on dihydropyridine receptor-ryanodine receptor (DHPR-RyR1) coupling by endogenous STIM1. The cytosolic Ca2+ level was also increased due to the reduction in SR Ca2+ level. In addition, R429C-expressing myotubes showed abnormalities in mitochondrial shape, a significant decrease in ATP levels, and the higher expression levels of mitochondrial fission-mediating proteins. Therefore, serial defects in SOCE, intracellular Ca2+ movement, and cytosolic Ca2+ level along with mitochondrial abnormalities in shape and ATP level could be a pathological mechanism of R429C for human skeletal muscular hypotonia. This study also suggests a novel clue that STIM1 in skeletal muscle could be related to mitochondria via regulating intra and extracellular Ca2+ movements.
Highlights
Excitation-contraction (EC) coupling is a prerequisite for skeletal muscle contraction[1,2,3]
In skeletal muscle (Fig. 1a), R429 of STIM1 to C (R429C) was expressed in mouse primary skeletal myotubes rather than in heterologous expression systems in order to avoid possible artefacts introduced by the cell system (Fig. 1b)
We found that R429C did not significantly affect the formation of skeletal myotubes during terminal differentiation (Fig. 1), suggesting that defects in myotube formations are not related to the human muscular hypotonia caused by R429C
Summary
Excitation-contraction (EC) coupling is a prerequisite for skeletal muscle contraction[1,2,3]. Ryanodine receptor 1 molecules (RyR1s, internal Ca2+ channels) on the membrane of the sarcoplasmic reticulum (SR, corresponding to ER in other cell types) are subsequently opened due to the physical interactions with the active DHPRs (i.e., DHPR-RyR1 coupling), which represents a key step in inducing intracellular Ca2+ movement that is required for skeletal muscle contraction. These interactions allow Ca2+ movement from the SR to the cytosol through RyR1s (i.e., intracellular Ca2+ movement). Considering that various mutations in STIM1 cause the human skeletal muscle diseases mentioned above, examining the pathological effect(s) of R429C on the major functions of skeletal muscle, such as intracellular Ca2+ movement, which is needed for skeletal muscle contraction, is important and helpful in understanding the multiple physiological roles of STIM1 in skeletal muscle
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