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Regulation of store-operated calcium entries and mitochondrial uptake by minidystrophin expression in cultured myotubes

A. Vandebrouck^*, T. Ducret, O. Basset, S. Sebille^*, G. Raymond^*, U. Ruegg, P. Gailly, C. Cognard^* and B. Constantin^*,1

^* Institut de Physiologie et Biologie Cellulaires, CNRS, UMR-6187, University of Poitiers, Poitiers, France ;
Laboratory of Pharmacology, University of Geneva, Geneva, Switzerland; and
Departement de Physiologie, Université Catholique de Louvain, Brussels, Belgium

¹Correspondance: Institut de Physiologie et Biologie Cellulaires, CNRS, UMR-6187, University of Poitiers, Poitiers 86022, France. E-mail: bruno.constantin{at}univ-poitiers.fr

SPECIFIC AIMS

The assumption that dystrophin could play a regulatory role on calcium homeostasis prompted us to determine whether a minidystrophin, a candidate for viral-mediated gene therapy, could play the same role. We have previously shown that forced expression of minidystrophin was able to restore normal calcium resting levels and transients in Sol8 myotubes. Calcium mishandling in dystrophic muscle cells could be related to increased calcium influx and more active store-operated calcium channels. The present study was performed in order to determine whether store-dependent calcium influx could be regulated by the presence of minidystrophin, as well as the cytosolic and mitochondrial Ca²⁺ transients. The store-dependent calcium entry (SOCE) was analyzed at 3 days of differentiation, before the appearance of calcium mishandling observed in more advanced dystrophin-deficient myotubes.

PRINCIPAL FINDINGS

1. Minidystrophin reduced calcium influxes induced by depletion of calcium stores from sarcoplasmic reticulum
Bath application of Mn²⁺ resulted in a progressive quench of fura-2 fluorescence emitted from myotubes after depletion of calcium stores by treatment with a SERCA blocker (Fig. 1 A). This allowed us to record cation transmembrane influx activated by depletion of sarcoplasmic reticulum calcium stores. The quench rate, reflecting the rate of Ca²⁺ entry, was 2-fold lower in myotubes expressing recombinant minidystrophin underneath the sarcolemma (Fig. 1B ). However, mini-dystrophin had no effect on store-dependent calcium influx in myoblasts where the protein was distributed throughout the cytosol. Iterative stimulation of calcium release by caffeine in the presence of a SERCA blocker also induced store-dependent calcium influx in myotubes. The rate of calcium entry was also higher in dystrophin-deficient myotubes (Fig. 1C , SolC1) than in myotubes expressing minidystrophin (Fig. 1C , SolD6), which displayed the same quench rates as normal myotubes expressing the native dystrophin (Fig. 1C , SolC57).

View larger version (25K): [in this window] [in a new window]   Figure 1. Effect of SR depletion on calcium transients in SolC1dys–, SolD7minidys+, SolD6minidys+ and SolC57dys+ myoblasts, and myotubes. A) Measurement of the fura-2 emission intensity (with an excitation at 360 nm) in representative SolD7minidys+ and SolC1dys– (dystrophin-deficient) myotubes. In the example illustrated here, Ca2+ stores had been depleted by application of thapsigargin (1 µM) in Ca2+ free recording medium. The progressive quenching of fluorescence is due to the influx of Mn2+ taken as a surrogate of Ca2+ after the reintroduction of 2 mM Ca2+ with 50 µM Mn2+ in the extracellular medium. B) Slope of the Mn2+-induced fluorescence quenching in SolC1dys– (SolC1dys– mb, n=6) and SolD7minidys+ (SolD7minidys+ mb, n=7) myoblasts and SolC1dys– (SolC1dys– MT, n=11) and SolD7minidys+ (SolD7minidys+ MT, n=10) myotubes. Insert: Immunostaining of minidystrophin in SolD7minidys+ myoblasts and myotubes at F+3 stage. Images were taken and analyzed by fluorescence confocal microscopy. C) Slope of the Mn2+-induced fluorescence quenching in SolC1dys– (SolC1dys– MT, n=49), SolD6minidys+ (SolD6minidys+ MT, n=37) and SolC57dys+ (SolC57dys+ MT, n=35) myotubes. Store were emptied with three stimulations by 10 mM caffeine in the presence of 5 µM CPA and the absence of Ca2+, space out by 15 µM CPA in Ca2+-free solution. Reintroduction of 1.8 mM Ca2+ with 50 µM Mn2+ in extracellular medium leads to a decrease of the fluorescence intensity corresponding to SOCE. Insert: Immunostaining of dystrophin in SolC57dys myotubes and of minidystrophin in SolD6minidys+ myotubes at F+3 stage. ***P < 0.001.

2. Minidystrophin reduced duration of store-dependent calcium transients in myotube cytosol
Changes in free cytosolic calcium concentration were determined by measuring the fluorescence ratio of the Ca²⁺ indicator Indo-1. Repetitive activation of calcium release and inhibition of SERCA induced store-dependent cytosolic calcium transients. Application of 50 µM or 10 µM 2-APB promoted a dual effect, which has been previously reported elsewhere to be a property of SOCE. Dystrophin-deficient myotubes displayed long-lasting calcium increase with slow decay. Minidystrophin expressing myotubes responded by more transitory cytosolic calcium elevation with the same kinetic parameters as in myotubes expressing native dystrophin.

3. The long-lasting store-dependent calcium transients in dystrophin-deficient myotubes were dependent on mitochondrial activity
Depolarization of mitochondrial potential by pretreatment with 2 µM of the protonophore FCCP decreased the amplitude of cytosolic calcium transients. In dystrophin-deficient myotubes this effect was accompanied by a decreased duration of the decay phase. It is known that greater calcium uptake by mitochondria can increase the size and duration of SOCE, by reducing the rate and extent of Ca²⁺-dependent slow inactivation. The facilitation by mitochondrial uptake of SOCE could explain the increase in duration of store-dependent calcium transients. We thus explored calcium transients in mitochondria after SOCE activation by store depletion.

4. Minidystrophin reduced the mitochondrial calcium uptake during activation of SOCE
Measurement of Ca²⁺ transients in mitochondria during store-operated influx was performed by using an aequorin targeted to mitochondria (mtAeq). Depletion of calcium stores also induced store-dependent calcium transients in mitochondria. Comparisons showed that these transients were significantly higher in dystrophin-deficient myotubes than when minidystrophin was expressed (Fig. 2 ). The rapid uptake of store-dependent calcium influx by mitochondria was already shown in other preparations. The present results demonstrate that reintroduction of minidystrophin in cultured myotubes is indeed able to reduce store-dependent calcium influx as well as mitochondrial uptake after SOCE activation. This could protect mitochondria from an exaggerated increase in calcium uptake during this process.

View larger version (24K): [in this window] [in a new window]   Figure 2. Measurement of intramitochondrial calcium variation during SOCE. Myotubes transiently expressed mtAEQ. A) Example of intramitochondrial calcium measurement. Stores were emptied with three stimulations by 10 mM caffeine in the presence of 5 µM CPA and the absence of Ca2+. Reintroduction of 1.8 mM Ca2+ in extracellular medium leads to an increase of [Ca2+]m corresponding to uptake of SOCE. Six parameters were measured on biphasic Ca2+ increase: 2 amplitudes (A1 and A2), 2 time-to-peak (T1 and T2) and 2 areas under the curve (Area1 and Area2). B) Bar chart comparing the amplitude (µM), the time-to-peak (s), and the area-under-curve of the increase of Ca2+ signal upon readmission of Ca2+ in SolC1dys– (black) and SolD7minidys+ (gray) . Numbers of cell (n) are: SolC1dys–, 19; SolD7minidys+, 27. C) Representative example of intramitochondrial Ca2+ signals during SOCE in SolC1dys– (black) and SolD7minidys+ (gray). Traces were normalized vs. the maximum of each peak amplitude. *P < 0.05.

CONCLUSIONS AND SIGNIFICANCE

Earlier single channel studies of mouse muscle fibers demonstrated that store-dependent calcium currents are more important in fibers from mdx dystrophic mice than from normal mice. These currents were shown to involve TRPC proteins. This suggests that in normal skeletal muscle cells, dystrophin could regulate the activity of store-operated channels, then attenuate capacitative calcium entries. Together, our data indicate that reintroduction of minidystrophin is able to reduce store-dependent calcium influx and restore normal rates of calcium entries similar to those of myotubes expressing native dystrophin. Regulation of sarcolemmal calcium influx was observed at 3 days of differentiation, only when minidystrophin was sorted at the sarcolemma. The increase in cytosolic calcium associated with this entry was shortened and, more important, the uptake of calcium by mitochondria was reduced. In dystrophin-deficient myotubes, the higher uptake of calcium by mitochondria in response to an increased store-dependent influx may result in prolonged activation of SOCE. This in turn increases the amount of calcium buffered by mitochondria. On the contrary, expression of minidystrophin decreases SOCE through the sarcolemma, which regulates the interplay between calcium entries, mitochondria, and sarcoplasmic reticulum (Fig. 3 ). Restoration by minidystrophin of normal calcium influx and the lower calcium uptake by mitochondria precede the protective effect against alteration of calcium homeostasis observed later. Modulation of SOCE by dystrophin and its consequences on mitochondria uptake could be key in explaining why the absence of this protein from the cortical cytoskeleton is able to alter calcium homeostasis during muscle cell activation. Dystrophin could control the sarcolemmal calcium entries in muscle cells by a complex mechanism involving protection against membrane ruptures and against overactivation of mechanosensitive channels and store-dependent channels. Our observations provide additional arguments for the use of minidystrophin for functional recovery of dystrophin-deficient muscles. The C-terminal domain was shown to be involved in the interaction with the dystrophin-associated complex or in the recovery of membrane associated NO synthase. One can speculate that this functional domain and the complex interactions with associated proteins are participating in the normal regulation of SOCE. Future investigations will be conducted to explore the molecular interaction of the dystrophin-associated complex with store-dependent channels. The present work reports a particularly clear example of functional involvement of a cytoskeletal protein assemblage in the modulation of ionic transfers through the surface and internal membranes of interacting compartments.

View larger version (53K): [in this window] [in a new window]   Figure 3. Schematic diagram. SOC, store-generated channel; RYR, ryanodine receptor; DHPR, dihydroxyuridine receptor; DGC, dystrophin-glycoprotein complex; SR, sarcoplasmic reticulum; ECM, extracellular matrix.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3633fje;

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This Article Full Text (PDF) All Versions of this Article: 20/1/136 04-3633fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vandebrouck, A. Articles by Constantin, B. Search for Related Content PubMed PubMed Citation Articles by Vandebrouck, A. Articles by Constantin, B.