Abstract
Store-operated Ca2+ entry (SOCE) contributes to Ca2+ handling in normal skeletal muscle function, as well as the progression of muscular dystrophy and sarcopenia, yet the mechanisms underlying the change in SOCE in these states remain unclear. Previously we showed that calsequestrin-1 (CSQ1) participated in retrograde regulation of SOCE in cultured skeletal myotubes. In this study, we used small-hairpin RNA to determine whether knockdown of CSQ1 in adult mouse skeletal muscle can influence SOCE activity and muscle function. Small-hairpin RNA against CSQ1 was introduced into flexor digitorum brevis muscles using electroporation. Transfected fibers were isolated for SOCE measurements using the Mn2+ fluorescence-quenching method. At room temperature, the SOCE induced by submaximal depletion of the SR Ca 2+ store was significantly enhanced in CSQ1-knockdown muscle fibers. When temperature of the bathing solution was increased to 39°C, CSQ1-knockdown muscle fibers displayed a significant increase in Ca2+ permeability across the surface membrane likely via the SOCE pathway, and a corresponding elevation in cytosolic Ca2+ as compared to control fibers. Preincubation with azumolene, an analog of dantrolene used for the treatment of malignant hyperthermia (MH), suppressed the elevated SOCE in CSQ1-knockdown fibers. Because the CSQ1-knockout mice develop similar MH phenotypes, this inhibitory effect of azumolene on SOCE suggests that elevated extracellular Ca2+ entry in skeletal muscle may be a key factor for the pathophysiological changes in intracellular Ca2+ signaling in MH.
| Original language | English |
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| Pages (from-to) | 1556-1564 |
| Number of pages | 9 |
| Journal | Biophysical Journal |
| Volume | 99 |
| Issue number | 5 |
| DOIs | |
| State | Published - Sep 8 2010 |
Funding
In this study, we show that knockdown of CSQ1 influences Ca 2+ signaling in adult skeletal muscle by perturbing Ca 2+ entry across the sarcolemmal/T-tubular membrane, which increases the vulnerability of the skeletal muscle to heat stress. The elevated rate of Mn 2+ -entry observed in the shCSQ1-treated FDB fibers can be blocked by azumolene to a level comparable to that of the shCON-treated fibers, indicating that reduced CSQ1 expression may affect RyR1-coupled SOCE activation in skeletal muscle. Taking into account that systemic knockout of CSQ1 in mice leads to hypersensitivity of the animal to heat and volatile anesthetics with a phenotype resembling that of the MH ( 15 ), our data provide further evidence that increased activity of RyR1-coupled SOCE could be associated with the etiology of MH. The therapeutic effect of dantrolene, an azumolene analog used for clinical treatment of MH, seems to act through a mechanism that involves the suppression of RyR1-activated SOCE. Operation of SOCE requires retrograde signaling from the ER/SR membrane to the sarcolemmal membrane, across a distance of ∼100 Å in the triad junction of skeletal muscle ( 26 ). Because CSQ1 is a Ca 2+ -binding protein located inside the SR lumen, changes in SOCE function associated with CSQ1 knockdown could reflect the following possibilities: 1), expression of the molecular components for SOCE function could be altered with reduced expression of CSQ1; 2), reduced CSQ1 expression likely alters the SR Ca 2+ release process and affects the retrograde steps of SOCE activation; and 3), knockdown CSQ1 expression could alter the conformational coupling between the SR and sarcolemmal membrane components and thus indirectly influence permeability of Ca 2+ across the sarcolemmal membrane. Our data provides some insights into these possibilities. First, it is well known that STIM1 located on the ER membrane functions as the luminal Ca 2+ sensor that controls opening of the Orai Ca 2+ channel located on the plasma membrane for operation of SOCE ( 25 ). The changes in SOCE function could be a result of altered expression of either STIM1 or Orai1. Our Western blot data showing that knockdown of CSQ1 expression leads to reduced expression of Orai1 is counterintuitive to the elevated SOCE activity in skeletal muscle. We speculate that this may represent a compensatory mechanism for the survival of skeletal muscle in adaptation to the reduced CSQ1 expression and elevated SOCE activity. At present, we do not know if the increased SOCE activity associated with CSQ1 knockdown is due to an increase in preformed STIM1-Orai1 complexes in the sarcolemma or T-tubule membrane that can increase the efficiency of signal transduction and Ca 2+ entry, or whether the expression of other Orai homologs, such as Orai2 and Orai3, is altered in skeletal muscle. Second, as CSQ1 is a SR-resident Ca 2+ buffering protein, altering expression level of CSQ1 would change the SR Ca 2+ buffering capacity and accessibility of the free Ca 2+ inside the SR. Previous studies showed that saponin-permeabilized CSQ1-null muscle fibers seem to have both a reduced response to electric stimulation and a smaller SR Ca 2+ content ( 14 ). Recent studies by Sztretye et al. ( 23 ) also found that the kinetics of voltage-induced Ca 2+ release and the evacuability of Ca 2+ from the SR store in response to sustained voltage stimulation are modified with reduced CSQ1 expression. Our data demonstrate that the caffeine-releasable SR Ca 2+ content was smaller and the Ca 2+ release kinetics faster with reduced CSQ1 expression, consistent with these studies. The tight link between SR Ca 2+ storage and SOCE activity has long been established ( 13 ), and it is conceivable that a reduced SR Ca 2+ store—possibly due to compromised Ca 2+ buffering capacity secondary to a reduced CSQ1 protein content in SR—leads to higher SOCE activity. In addition, the faster kinetics of SR Ca 2+ release in the shCSQ1-treated muscle fiber could contribute to the elevated SOCE, possibly through enhanced clustering of STIM1 and Orai1, to excessively activate SOCE at the plasma membrane. Third, it is well known that close conformational coupling between plasma membrane and ER membrane constituents is essential for efficient operation of SOCE ( 10,22 ) and graded activation of SOCE has been shown to couple to graded reduction of the SR Ca 2+ store ( 10,13 ). Indeed, ultrastructural adaptations have been shown to occur in CSQ1 null muscle fibers ( 14 ), providing indirect evidence for the possibility of change in conformational coupling between the two membrane compartments in the absence of CSQ1 protein. In addition, a component of SOCE in skeletal muscle has been shown to involve conformational changes in the RyR1 ( 13 ). In this study, several lines of evidence imply that alteration in the conformational coupling might act synergistically with the altered SR Ca 2+ release kinetics to activate SOCE in CSQ1-knockdown skeletal muscle. Here, we show that azumolene suppresses the elevated SOCE activity in CSQ1 knockdown muscle fibers. This drug has been shown to uncouple the tight link between SR Ca 2+ depletion via RyR1 and the activation of SOCE ( 13 ). We also show that muscle fibers with reduced CSQ1 expression display increased vulnerability to heat-induced changes in Ca 2+ permeability across the sarcolemmal membrane without substantial depletion of the SR Ca 2+ store, pointing to the possibility of conformational changes in the SOCE machinery. The steady-state resting Ca 2+ levels do not show significant differences between the shCSQ1 and shCON muscle fibers at ambient room temperature, suggesting that integrity of the control mechanism for movement of extracellular Ca 2+ across the sarcolemmal membrane is not likely to be altered with reduced CSQ1 expression in skeletal muscle under nonstressed condition. A sharp difference between the shCSQ1 and shCON muscle fibers is revealed in divalent cation permeability across the sarcolemmal membrane and cytosolic Ca 2+ at elevated temperatures. Previous work by van der Poel and Stephenson ( 27 ) showed that increased temperature to 40°C led to increased SR Ca 2+ leakage in mechanically skinned muscle fibers, presumably through temperature-induced superoxide production. Our data show that in addition to its effect on the SR Ca 2+ transport process, increase in temperature likely has a separate effect on SOCE, because the inhibition of Ca 2+ entry from the extracellular space by Ni 2+ eliminates the difference in [Ca 2+ ] i transients in shCON and shCSQ1-fibers ( Fig. 4 ). Furthermore, although exposure of muscle fibers to higher temperatures may activate heat-sensitive Ca 2+ entry into the cytosol, coordinated action of SERCA-mediated Ca 2+ uptake into the SR (due to its high Q10 value) and Na + /Ca 2+ exchanger- or plasma membrane Ca 2+ -ATPase-mediated Ca 2+ extrusion mechanisms allows for tight control of Ca 2+ homeostasis in the native skeletal muscle. This may explain the minimal effect of Ni 2+ on the resting [Ca 2+ ] i in shCON-treated muscle fibers at 39°C. Nevertheless, the clear effect of Ni 2+ on the elevated [Ca 2+ ] i and inhibition of azumolene on Mn 2+ -quenching of FURA-2 fluorescence at 39°C in shCSQ1-tranfected muscle fibers suggest that a significant component of the defective [Ca 2+ ] i homeostasis under high temperature may be due to the elevated RyR1-coupled SOCE seen after knockdown of CSQ1. Malignant hyperthermia is a skeletal muscle syndrome associated with anesthetic-triggered increases in [Ca 2+ ] i and excessive production of body heat ( 17 ). Although many studies show that a disturbance of Ca 2+ release from SR is associated with triggering an MH crisis, several recent studies have begun to reveal the contribution of SOCE to muscle physiology and disease. With regard to MH, in particular, elevated SOCE could be another pathway for the uncontrolled rise in cytoplasmic [Ca 2+ ] associated with MH. Our data presented here add further insights into the physiological roles of CSQ1 in normal muscle function and its potential for dysfunction during disease states. The parallel changes of heat-sensitivity in CSQ1 knockdown fibers and susceptibility of CSQ1-null mice to MH-like episodes further suggest that CSQ1 could potentially participate in the etiology of MH. In addition, because many genetic mutations in CSQ1 are linked to skeletal muscle or cardiac dysfunction in human diseases, our data could have broad implications for clinical medicine. Targeting SOCE or CSQ1 could be a potential treatment of MH and/or the Ca 2+ -dependent pathology of the dystrophinopathies and sarcoglycanopathies ( 28 ). We thank Dr. Jingsong Zhou for help with the single-muscle-fiber Western-blot technique. This work was supported by grants from the National Institutes of Health (Bethesda, MD) to J.M. and N.W., a grant from the Korean Science and Engineering Foundation to D.H.K., a Scientist Development Award from the American Heart Association to X.Z., and a grant from the National Science Foundation of China to J.M. J.P. was supported by clinical funds of the Department of Anesthesiology, University of Pittsburgh School of Medicine.
| Funders |
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| Korean Science and Engineering Foundation |
| National Institutes of Health (NIH) |
| American the American Heart Association |
| Swanson School of Engineering, University of Pittsburgh |
| National Natural Science Foundation of China (NSFC) |
ASJC Scopus subject areas
- Biophysics