Abstract
Our previous work on human myoblasts suggested that a hyperpolarization followed by a rise in [Ca(2+)](in) involving store-operated Ca(2+) entry (SOCE) channels induced myoblast differentiation. Advances in the understanding of the SOCE pathway led us to examine more precisely its role in post-natal human myoblast differentiation. We found that SOCE orchestrated by STIM1, the endoplasmic reticulum Ca(2+) sensor activating Orai Ca(2+) channels, is crucial. Silencing STIM1, Orai1, or Orai3 reduced SOCE amplitude and myoblast differentiation, whereas Orai2 knockdown had no effect. Conversely, overexpression of STIM1 with Orai1 increased SOCE and accelerated myoblast differentiation. STIM1 or Orai1 silencing decreased resting [Ca(2+)](in) and intracellular Ca(2+) store content, but correction of these parameters did not rescue myoblast differentiation. Remarkably, SOCE amplitude correlated linearly with the expression of two early markers of myoblast differentiation, MEF2 and myogenin, regardless of the STIM or Orai isoform that was silenced. Unexpectedly, we found that the hyperpolarization also depends on SOCE, placing SOCE upstream of K(+) channel activation in the signaling cascade that controls myoblast differentiation. These findings indicate that STIM1 and Orai1 are key molecules for the induction of human myoblast differentiation.
Highlights
The molecular machinery that orchestrates storeoperated Ca2؉ entry (SOCE) has been recently discovered, improving our understanding of the cellular events connecting Ca2ϩ store depletion to SOCE activation [6]
Myoblasts were incubated for 5 h with a Small Interference RNA (siRNA) designed against STIM1 and 42 h later switched from a proliferation to a differentiation medium to induce their differentiation into multinucleated myotubes
Quantification of the MEF2/DAPI ratio yielded an index of 7 Ϯ 5% (n ϭ 7) instead of the control-siMed index of 60 Ϯ 18% (n ϭ 7), indicating that myoblast differentiation was severely impaired by the genetic invalidation of STIM1
Summary
Cell Culture—Muscle samples, cell dissociation, and clonal culture from satellite cells were prepared as previously described [1, 29, 30]. Blots were incubated with the primary antibodies diluted in T-TBS and nonfat milk as follows: mouse monoclonal antibody anti-GOK/ STIM1 1:500 (BD Biosciences) or rabbit polyclonal anti-STIM2 antibody (1:500; ProSci Inc.) and mouse monoclonal antibody against ␣-tubulin (clone DM1A, Sigma) 1:10,000. Image acquisition and analysis were carried out with the Metafluor 6.3r7 software (Molecular Devices, Visitron Systems GmbH, Puchheim, Germany). Rmax was evaluated in all conditions (using a solution containing 4 M ionomycin and 5 mM Ca2ϩ), ruling out any possible nonlinearity between data obtained in myoblasts transfected with different plasmids or siRNA. Confocal Monitoring of Membrane Potential—As described previously in Konig et al [2], confocal image acquisition of the fluorescent probe for membrane potential bis(1,3-dibarbituric acid)-trimethine oxonol (DiBAC4(3); Molecular Probes) was performed using a spinning disc confocal microscope. With [Naϩ]i ϩ [Kϩ]i assumed to be 150 mM, T ϭ 310 K, Z is the number of elementary charges of the ion, and F is the Faraday constant
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