STIM1 negatively regulates Ca2+ release from the sarcoplasmic reticulum in skeletal myotubes

Abstract

STIM1 (stromal interaction molecule 1) mediates SOCE (store-operated Ca²⁺ entry) in skeletal muscle. However, the direct role(s) of STIM1 in skeletal muscle, such as Ca²⁺ release from the SR (sarcoplasmic reticulum) for muscle contraction, have not been identified. The times required for the maximal expression of endogenous STIM1 or Orai1, or for the appearance of puncta during the differentiation of mouse primary skeletal myoblasts to myotubes, were all different, and the formation of puncta was detected with no stimulus during differentiation, suggesting that, in skeletal muscle, the formation of puncta is a part of the differentiation. Wild-type STIM1 and two STIM1 mutants (Triple mutant, missing Ca²⁺-sensing residues but possessing the intact C-terminus; and E136X, missing the C-terminus) were overexpressed in the myotubes. The wild-type STIM1 increased SOCE, whereas neither mutant had an effect on SOCE. It was interesting that increases in the formation of puncta were observed in the Triple mutant as well as in wild-type STIM1, suggesting that SOCE-irrelevant puncta could exist in skeletal muscle. On the other hand, overexpression of wild-type or Triple mutant, but not E136X, attenuated Ca²⁺ releases from the SR in response to KCl [evoking ECC (excitation-contraction coupling) via activating DHPR (dihydropyridine receptor)] in a dominant-negative manner. The attenuation was removed by STIM1 knockdown, and STIM1 was co-immunoprecipitated with DHRP in a Ca²⁺-independent manner. These results suggest that STIM1 negatively regulates Ca²⁺ release from the SR through the direct interaction of the STIM1 C-terminus with DHPR, and that STIM1 is involved in both ECC and SOCE in skeletal muscle.

Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass

“…In aortic endothelial cells, Stim1 potentiates IP3R-mediated ER Ca 2+ release (33). In muscle cells, Stim1 inhibits Ca 2+ discharge from the sarcoplasmic reticulum (34). Differences in ER Ca 2+ release were also noted in Stim1 −/− mast cells in response to antigen or Tg (35).…”

The Maintenance Ability and Ca 2+ Availability of Skeletal Muscle are Enhanced by Sildenafil

“…18,[30][31][32][33] Briefly, primary myoblasts were cultured on 10-cm plates coated with collagen in growth medium (F-10 Nutrient Mixture (HyClone Laboratories, Logan, UT, USA) containing 20% fetal bovine serum (FBS), 100 units ml − 1 penicillin, 100 μg ml − 1 streptomycin, 2 mM L-glutamine and 20 nM basic FGF) at 37°C in a 5% CO 2 incubator. For the differentiation of myoblasts into myotubes, myoblasts were replated either on 10-cm plates (for the quantitative RT-PCR (qPCR) analysis or for the preparation of myotube lysate) or on 96-well plates (for the single myotube Ca 2+ imaging experiment) coated with Matrigel (BD Biosciences, San Jose, CA, USA).…”

“…Primary skeletal myotubes were solubilized as previously described, 18,[30][31][32][33] and the solubilized lysate (10 μg of total protein) was subjected to SDS-PAGE (8, 10 or 12% gel). The proteins on the gel were transferred to a polyvinylidene difluoride membrane at 100 V for 1 h. The membranes were blocked with 5% (w/v) non-fat milk dissolved in PBS, incubated with a corresponding primary antibody, …”

“…Measurements of the width in primary skeletal myotubes (one criterion that is used to evaluate the degree of skeletal myotube formation (that is, the degree of differentiation) 18,[30][31][32] ) were performed as previously described. 30,31 The width at the thickest part of each myotube was measured using the ImageJ program (http://imagej.nih.gov/ij/).…”

“…30,31 The width at the thickest part of each myotube was measured using the ImageJ program (http://imagej.nih.gov/ij/).…”