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First published online 22 August 2006
doi: 10.1242/jcs.03184

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Ca²⁺-independent phospholipase A₂ enhances store-operated Ca²⁺ entry in dystrophic skeletal muscle fibers

Laboratory of Pharmacology, Geneva-Lausanne School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1211 Geneva 4, Switzerland

^* Author for correspondence (e-mail: Urs.Ruegg{at}pharm.unige.ch)

Accepted 18 July 2006

	Summary

Top Summary Introduction Results Discussion Materials and Methods References

 
Duchenne muscular dystrophy is caused by deficiency of dystrophin and leads to progressive weakness. It has been proposed that the muscle degeneration occurring in this disease is caused by increased Ca²⁺ influx due to enhanced activity of cationic channels that are activated either by stretch of the plasma membrane (stretch-activated channels) or by Ca²⁺-store depletion (store-operated channels). Using both cytosolic Ca²⁺ measurements with Fura-2 and the manganese quench method, we show here that store-operated Ca²⁺ entry is greatly enhanced in dystrophic skeletal flexor digitorum brevis fibers isolated from mdx^5cv mice, a mouse model of Duchenne muscular dystrophy. Moreover, we show for the first time that store-operated Ca²⁺ entry in these fibers is under the control of the Ca²⁺-independent phospholipase A₂ and that the exaggerated Ca²⁺ influx can be completely attenuated by inhibitors of this enzyme. Enhanced store-operated Ca²⁺ entry in dystrophic fibers is likely to be due to a near twofold overexpression of Ca²⁺-independent phospholipase A₂. The Ca²⁺-independent phospholipase A₂ pathway therefore appears as an attractive target to reduce excessive Ca²⁺ influx and subsequent degeneration occurring in dystrophic fibers.

Key words: Dystrophic skeletal muscle fibers, Store-operated channel, Ca²⁺-independent phospholipase A₂

	Introduction

Top Summary Introduction Results Discussion Materials and Methods References

 
Duchenne muscular dystrophy (DMD) is an X-linked hereditary disease affecting 1 in 3500 male births. DMD is caused by a mutation in the p21 region of the X chromosome, which leads to a deficiency of dystrophin, a 427 kDa protein located in the inner face of the plasma membrane (Blake et al., 2002). In normal skeletal muscle fibers, dystrophin is linked to cytoskeletal actin and plasma membrane glycoproteins associated with the extracellular matrix. Dystrophin provides a link between the cytoskeleton and the extracellular matrix and its absence in dystrophic fibers leads to progressive weakness and muscle degeneration (Blake et al., 2002). Muscle degeneration occurring in DMD has been proposed to be due to increased Ca²⁺ influx and abnormal Ca²⁺ homeostasis, resulting in increased proteolysis and mitochondrial Ca²⁺ overload (Alderton and Steinhardt, 2000; Basset et al., 2004; Gailly, 2002; Robert et al., 2001; Ruegg et al., 2002; Vandebrouck et al., 2006). Mitochondrial Ca²⁺ overload occurring in dystrophic muscle may lead to cytochrome C release and apoptosis (Basset et al., 2006; Duchen, 2004). The increased Ca²⁺ permeability of dystrophic fibers sarcolemma appears to be due to enhanced activity of non-selective cationic channels, which are activated either by Ca²⁺-store depletion (store-operated channels: SOC) or by stretch of the plasma membrane (Allen et al., 2005; Vandebrouck et al., 2006; Vandebrouck et al., 2002; Yeung and Allen, 2004; Yeung et al., 2004). Furthermore, it has been recently proposed that channels responsible for the enhanced Ca²⁺ influx in dystrophic fibers belong to the TRP family (Allen et al., 2005; Iwata et al., 2003; Maroto et al., 2005; Vandebrouck et al., 2002).

Three models have been put forward to explain the link between the sarco-endoplasmic Ca²⁺ store and SOC, also called `capacitative Ca<sup>2+</sup> entry' (Parekh and Putney, Jr, 2005). The first model postulates that SOC located in the plasma membrane may be activated by a conformational coupling with the channels responsible for Ca<sup>2+</sup> release, i.e. ryanodine and/or inositol 1,4,5-trisphosphate receptors (Kiselyov et al., 1998; Kiselyov et al., 2000). The second model proposes that capacitative Ca<sup>2+</sup> entry is activated by docking or fusion of secretory vesicles containing SOC activators (or channels) with the plasma membrane (Parekh and Putney, Jr, 2005). Along these lines, STIM1, a recently discovered transmembrane protein, is a Ca<sup>2+</sup> sensor activating SOC by migrating from the Ca<sup>2+</sup> store to the plasma membrane (Zhang et al., 2005). Finally, the third model postulates that a diffusible messenger termed `calcium influx factor' (CIF), most likely a phospholipid, is produced upon Ca²⁺-store depletion and activates SOC (Randriamampita and Tsien, 1993; Rzigalinski et al., 1999).

New evidence in favor of the idea of a diffusible messenger (such as CIF) produced by the sarcoplasmic reticulum (SR) upon Ca²⁺-store depletion was recently obtained (Smani et al., 2004; Smani et al., 2003; Vanden Abeele et al., 2004). It was further demonstrated that CIF can trigger SOC opening, but it is not clear if CIF activates SOC directly or if it involves additional mechanisms (Bolotina and Csutora, 2005; Trepakova et al., 2000). Several reports suggest that CIF acts through the stimulation of a Ca²⁺-independent PLA₂ (iPLA₂) (Smani et al., 2004; Smani et al., 2003; Vanden Abeele et al., 2004).

PLA₂ are enzymes that catalyze the hydrolysis of fatty acid ester bonds at the second position of diacyl-glycerophospholipids (Chakraborti, 2003) leading to the release of a fatty acid (among which arachidonic acid) and lysophospholipids. Isoforms of PLA₂ are distinguished according to their molecular mass, location and sensitivity to Ca²⁺. Cytosolic PLA₂ are Ca²⁺-dependent but the iPLA₂ family does not need Ca²⁺ for activation (Chakraborti, 2003). The involvement of PLA₂ isoforms in capacitative Ca²⁺ entry has been demonstrated in several types of cells (Hichami et al., 2002; Martinez and Moreno, 2005; Rzigalinski et al., 1999; Smani et al., 2003; Vanden Abeele et al., 2004; Zablocki et al., 2000). CIF may act through the inhibition of calmodulin binding to iPLA₂, resulting in iPLA₂ stimulation (Smani et al., 2004; Vanden Abeele et al., 2004). The resulting PLA₂ hydrolysis products would then be responsible for the stimulation of SOC (Smani et al., 2004). Indeed, it has been demonstrated that lysophospholipids or arachidonic acid and some metabolites of the latter can activate cationic channels such as SOC (Rzigalinski et al., 1999; Smani et al., 2004; So et al., 2005; Terasawa et al., 2002; Watanabe et al., 2003).

In dystrophic muscle, SOC have been proposed to be involved in enhanced Ca²⁺ influx (Vandebrouck et al., 2002). However, their regulation has not been studied so far. Here we have investigated the involvement of the PLA₂ pathway in the regulation of Ca²⁺ entry in both normal and dystrophic fibers isolated from murine flexor digitorum brevis (FDB) muscles.

Using both Ca²⁺ imaging and the manganese quench method, we show that store-operated Ca²⁺ entry is greatly enhanced in dystrophic fibers. We also demonstrate that this Ca²⁺ entry is controlled by iPLA₂ and that exaggerated Ca²⁺ influx occurring in dystrophic fibers can be attenuated by iPLA₂ inhibitors to a value close to the one of normal fibers. Finally, western blot analysis of muscle extracts revealed an increased expression of iPLA₂ in dystrophic muscles, suggesting that enhanced store-operated Ca²⁺ entry occurring in dystrophic fibers may be due, at least in part, to overexpression of iPLA₂. The iPLA₂ pathway therefore appears to be an attractive target to reduce excessive Ca²⁺ influx and subsequent degeneration occurring in dystrophic fibers.

	Results

Top Summary Introduction Results Discussion Materials and Methods References

 
In this study we have used Ca²⁺ imaging to investigate the regulation of store-operated Ca²⁺ entry in both normal (C57BL/6J) and dystrophic (mdx^5cv) isolated FDB fibers. In all experiments fibers were incubated with 30 µM BTS to inhibit contractions that may cause artifacts in Ca²⁺ measurements (Cheung et al., 2002). Because Ca²⁺ influx may occur through L-type voltage-gated Ca²⁺ channels during stimulation of fibers, experiments were performed in the presence of 1 µM nifedipine, a specific blocker of L-type voltage-gated Ca²⁺ channels in skeletal muscle (O'Connell and Dirksen, 2000).

Ca²⁺ transients triggered by KCl depolarizations in normal and dystrophic fibers
In skeletal muscle, depolarization of the plasma membrane triggers activation of L-type voltage-gated Ca²⁺ channels, allosterically activating Ca²⁺ release through the opening of ryanodine receptors (Berchtold et al., 2000).

Fig. 1A,B shows typical Ca²⁺ transients triggered by KCl depolarizations in C57BL/6J and mdx^5cv FDB fibers. Basal cytosolic Ca²⁺ concentrations and peak Ca²⁺ concentrations following KCl depolarization were found to be very similar in normal and dystrophic fibers [56.5±1.6 nM (n=87) and 60.7±1.3 nM (n=90); 563.4±39.8 nM (n=87) and 629.4±46.5 nM (n=90), respectively; Fig. 1C]. Similar results were obtained following nicotinic receptor activation with acetylcholine or ryanodine receptor stimulation with caffeine (Fig. 7A).

View larger version (34K): [in this window] [in a new window]   Fig. 1. KCl-induced cytosolic Ca2+ transients in isolated FDB fibers from C57BL/6J and mdx5cv mice. (A,B) Calcium transients evoked by pressure ejection of a high KCl solution in isolated C57BL/6J and mdx5cv fibers, respectively. Top panels in A and B show time series of pseudocolor F340/F380 ratio images of isolated C57BL/6J and mdx5cv fibers, respectively. Bottom panels represent global cytosolic Ca2+ transients triggered by high-KCl calculated from the whole fiber perimeter. Red circles on curves correspond to recording time of the 18 ratio images in both cases. (C) Plot of average [Ca2+]i at the base line (b) and at the peak of KCl-induced Ca2+ transients (p) for both C57BL/6J and mdx5cv fibers. (D) Plot of average half time of decay of KCl-induced Ca2+ transients in both C57BL/6J and mdx5cv fibers analyzed in C. Numbers of fibers tested (from six mice for C57BL/6J fibers and from 10 mice for mdx5cv fibers) are indicated on bar graphs.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Effect of BTP2 and BEL on caffeine-induced Mn2+ entry in C57BL/6J and mdx5cv fibers. (A) Left panel: Ca2+ transients triggered by caffeine (50 mM) in C57BL/6J and mdx5cv fibers. Right panel: plot of average [Ca2+]i at the base line (b) and at the peak of caffeine-induced Ca2+ transients (p) for both C57BL/6J and mdx5cv fibers. (B) Effect of BTP2 (5 µM) and BEL (5 µM) on Mn2+ entry triggered by caffeine in C57BL/6J and mdx5cv fibers. Prior to caffeine application, fibers were incubated with a solution containing 1 mM MnCl2 and 1.7 mM CaCl2. On the same graph and for each type of fibers are shown results from a control experiment and from experiments performed on fibers preincubated with either BTP2 (5 µM) for 10 minutes or BEL (5 µM) for 20 minutes (hatched trace). As caffeine triggered small increase in Fura-2 fluorescence even when excitation was set to 360 nm, maximal fluorescence was set to 100% and fluorescence quench was measured 90 seconds after caffeine application. (C) Compiled data showing the percentage of fluorescence decrease 90 seconds after caffeine application in control condition and when fibers had been preincubated with either BTP2 or BEL. Vertical hatched line on recordings shown in B indicates the time for measurements of fluorescence decrease. Numbers of fibers tested (from four C57BL/6J mice and four mdx5cv mice) are indicated on bar graphs.

However, kinetic properties of KCl-induced Ca²⁺ transients were altered in mdx^5cv fibers compared with C57BL/6J fibers. Indeed, the decay phase of KCl-induced Ca²⁺ transients was slowed down in mdx^5cv fibers, as measured by the increased half time of decay in dystrophic fibers as compared with normal fibers (3.03±0.25 seconds, n=90 and 2.22±0.09 seconds, n=87, respectively; Fig. 1D).

KCl-induced Ca²⁺ transients in the absence of extracellular Ca²⁺
Ca²⁺ transients triggered by KCl depolarizations were recorded in Ca²⁺-free solution in both types of fibers, indicating that Ca²⁺ influx is not needed to trigger Ca²⁺ release through ryanodine receptors, as previously reported for skeletal-type excitation-contraction coupling (Lamb, 2002; O'Brien et al., 2002).

Half times of decay of KCl-induced Ca²⁺ transients were significantly reduced for both types of fibers in Ca²⁺-free solution containing 1 mM EGTA (from 2.47±0.13 seconds to 1.34±0.12 seconds and from 3.14±0.74 seconds to 1.27±0.12 seconds for C57BL/6J and mdx^5cv fibers, respectively; Fig. 2A,C), with no significant effect on peak Ca²⁺ transients in both cases (Fig. 2B). Experiments conducted on single mdx^5cv fibers also showed that the decay phase of KCl-induced Ca²⁺ transients was greatly increased when Ca²⁺ was present in the extracellular medium (Fig. 2D). These results indicate that Ca²⁺ influx took place during KCl-induced Ca²⁺ transients and also that this is the primary trigger responsible for the slow decay phase of KCl-induced Ca²⁺ transients in both types of fibers.

View larger version (36K): [in this window] [in a new window]   Fig. 2. KCl-induced cytosolic Ca2+ transients in isolated FDB fibers from C57BL/6J and mdx5cv mice recorded in Ca2+-free solution. (A) Cytosolic Ca2+ transients recorded in C57BL/6J and mdx5cv fibers in control conditions and in Ca2+-free solution containing 1 mM EGTA. (B) Plot of average [Ca2+]i at the base line (b) and at the peak of KCl-induced Ca2+ transients (p) for both C57BL/6J and mdx5cv fibers in control conditions and in Ca2+-free solution containing 1 mM EGTA. (C) Plot of average half time of decay of high KCl-induced Ca2+ transients in both C57BL/6J and mdx5cv fibers in control condition and in Ca2+-free solution containing 1 mM EGTA. Data come from the fibers analyzed in B. (D) A single mdx5cv fiber was incubated with Ca2+-free solution containing 1 mM EGTA and KCl-induced Ca2+ transient was recorded. After 10 minutes, Ca2+-free solution was replaced with a solution containing 1.7 mM Ca2+, and KCl-induced Ca2+ transient was recorded. On the left panel, Ca2+ response in Ca2+-free solution is superimposed to Ca2+ response recorded in Ca2+-containing solution. Numbers of fibers tested (from 2 C57BL/6J mice and 2 mdx5cv mice) are indicated on bar graphs.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Effect of the store-operated channel blocker BTP2 on KCl-induced cytosolic Ca2+ transients in isolated FDB fibers from C57BL/6J and mdx5cv mice. (A) KCl-induced cytosolic Ca2+transients in isolated FDB fibers from C57BL/6J and mdx5cv mice in control conditions and after preincubation of fibers with 5 µM BTP2 for 10 minutes. (B) Plot of average [Ca2+]i at the base line (b) and at the peak of KCl-induced Ca2+ transients (p) for both C57BL/6J and mdx5cv fibers in control conditions and in the presence of 5 µM BTP2. (C) Plot of average half time of decay of high KCl-induced Ca2+ transients in both C57BL/6J and mdx5cv fibers in control condition and in the presence of 5 µM BTP2. Data correspond to the fibers analyzed in B. Numbers of fibers tested (from two C57BL/6J mice and four mdx5cv mice) are indicated on bar graphs.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Effect of the PLA2 inhibitor AACOCF3 on KCl-induced cytosolic Ca2+ transients in isolated FDB fibers from C57BL/6J and mdx5cv mice. (A) KCl-induced cytosolic Ca2+ transients in isolated FDB fibers from C57BL/6J and mdx5cv mice in control conditions and after preincubation of fibers with 50 µM AACOCF3 for 10 minutes. (B) Plot of average [Ca2+]i at the base line (b) and at the peak of KCl-induced Ca2+ transients (p) for both C57BL/6J and mdx5cv fibers in control condition and in the presence of 50 µM AACOCF3. (C) Plot of average half time of decay of KCl-induced Ca2+ transients in both types of fibers without and with 50 µM AACOCF3. Data correspond to the fibers analyzed in B. Numbers of fibers tested (from two C57BL/6J mice and three mdx5cv mice) are indicated on bar graphs.

Since AACOCF₃ is known to inhibit Ca²⁺-dependent PLA₂ (cPLA₂) as well as Ca²⁺-independent PLA₂ (iPLA₂) (Ackermann et al., 1995), we next tested the effect of the specific iPLA₂ inhibitor bromoenol lactone (BEL) (Smani et al., 2003). Half time of decay of KCl-induced Ca²⁺ transients recorded in mdx^5cv fibers was significantly reduced when cells had been treated with 5 µM BEL (from 3.05±0.37 seconds to 1.94±0.15 seconds for control and BEL treated fibers, respectively; Fig. 5A,C), suggesting that iPLA₂ may be involved in the regulation of store-operated Ca²⁺ influx occurring during the late phase of KCl-induced Ca²⁺ responses in dystrophic fibers. Consistent with the data obtained with AACOCF₃, pretreatment with BEL did not have a significant effect on the decay phase of KCl-induced Ca²⁺ transient in normal fibers (half times of decay were 2.48±0.18 seconds and 2.04±0.22 seconds for control and BEL-treated fibers, respectively; Fig. 5C). With both types of fibers, pretreatment with BEL slightly decreased peak Ca²⁺ transients (Fig. 5B). Similar experiments were conducted in mdx^5cv fibers stimulated with 100 µM acetylcholine (ACh). As shown in Fig. 5D, half times of decay of ACh-induced Ca²⁺ transients were significantly reduced in BEL pretreated fibers (from 4.17±0.50 seconds to 2.77±0.35 seconds in control and BEL-treated fibers, respectively). These results suggest that iPLA₂ may be involved in the regulation of store-operated Ca²⁺ influx occurring during a Ca²⁺ transient in mdx^5cv fibers stimulated by either KCl depolarization or nicotinic receptor activation.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Effect of the iPLA2 inhibitor BEL on KCl-induced cytosolic Ca2+ transients in isolated FDB fibers from C57BL/6J and mdx5cv mice. (A) KCl-induced cytosolic Ca2+ transients in isolated FDB fibers from C57BL/6J and mdx5cv mice in control conditions and after preincubation of fibers with 5 µM BEL for 20 minutes (B) Plot of average [Ca2+]i at the base line (b) and at the peak of KCl-induced Ca2+ transients (p) for both C57BL/6J and mdx5cv fibers in control condition and after preincubation of fibers with 5 µM BEL. (C) Plot of average half times of decay of KCl-induced Ca2+ transients in both C57BL/6J and mdx5cv fibers in control condition and after preincubation of fibers with 5 µM BEL. Data correspond to the fibers analyzed in B. (D) Acetylcholine (ACh)-induced cytosolic Ca2+ transients in isolated mdx5cv FDB fibers in control condition and in fiber pretreated with 5 µM BEL for 20 minutes. Right panel: average half time of decay of ACh-induced Ca2+ transients in control and BEL-treated fibers. Numbers of fibers tested (from two C57BL/6J mice and three mdx5cv mice) are indicated on bar graphs.

Effect of BTP2 and BEL on Ca²⁺ entry triggered by Ca²⁺-store depletion
To investigate more directly the possible involvement of iPLA₂ in store-operated Ca²⁺ entry triggered by Ca²⁺-store depletion and also to compare store-operated Ca²⁺ entry in normal and dystrophin-deficient FDB fibers, we preincubated fibers in Ca²⁺-free solution containing 1 µM thapsigargin, a potent inhibitor of the SR Ca²⁺ ATPase (SERCA), to deplete SR Ca²⁺ stores. Ca²⁺ re-addition triggered an immediate Ca²⁺ increase in both C57BL/6J and mdx^5cv fibers, as expected when SERCA is blocked (Fig. 6A). When both types of fibers were preincubated with 5 µM BTP2 or 10 µM Gd³⁺, a well known SOC blocker (Allen et al., 2005), Ca²⁺ responses triggered by Ca²⁺ re-addition were nearly completely blocked, showing that they are due to Ca²⁺ influx through SOC (Fig. 6B,D). Ca²⁺ increases triggered by Ca²⁺ re-addition in SR-depleted fibers were 1100±161.6 nM and 402.6±51.9 nM in mdx^5cv and C57BL/6J fibers, respectively (Fig. 6A,D), indicating that store-operated Ca²⁺ influx is enhanced about 2.5-fold in mdx^5cv fibers compared with C57BL/6J fibers.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Store-operated Ca2+ entry in C57BL/6J and mdx5cv fibers and effect of the iPLA2 inhibitor BEL. Both types of fibers were preincubated with thapsigargin (1 µM) for 10 minutes in Ca2+-free solution prior to calcium re-addition (2 mM), leading to store-operated Ca2+ entry. (A) Ca2+ increases recorded in C57BL/6J and mdx5cv fibers following preincubation with thapsigargin and Ca2+ re-addition. (B) Effect of the store-operated channel blocker BTP2 (5 µM, 10 minutes preincubation) on Ca2+ increases triggered by Ca2+ re-addition in both C57BL/6J and mdx5cv fibers treated with thapsigargin. (C) Effect of the iPLA2 inhibitor BEL (5 µM, 20 minutes preincubation) on Ca2+ increases triggered by Ca2+ re-addition in both C57BL/6J and mdx5cv fibers treated with thapsigargin. (D) Average values showing the effect of BTP2, Gd3+ and BEL on store-operated Ca2+ entry in both types of fibers. Numbers of fibers tested (from five C57BL/6J mice and four mdx5cv mice) are indicated on bar graphs.

To investigate the possible involvement of iPLA₂ in store-operated Ca²⁺ entry, fibers were treated with 5 µM BEL and then tested with a similar protocol. Results indicate that BEL significantly reduced store-operated Ca²⁺ entry in mdx^5cv fibers (1100±161.6 nM to 488.4±119 nM for control and BEL treated cells, respectively; Fig. 6C,D). However, BEL had little effect in C57BL/6J fibers (402.6±51.9 nM to 267.7±69.9 nM for control and BEL-treated cells, respectively; Fig. 6C,D). Altogether these results indicate that iPLA₂ is involved in the regulation of store-operated Ca²⁺ influx triggered by SR Ca²⁺-store depletion in mdx^5cv fibers.

Effect of BTP2 and BEL on caffeine-induced Mn²⁺ entry
Thapsigargin is known to trigger an almost complete Ca²⁺-store depletion. To investigate whether iPLA₂ is involved in SOC regulation for lower levels of Ca²⁺-store depletion, we investigated the effect of iPLA₂ inhibition on caffeine-induced Mn²⁺ entry. Caffeine is known to trigger Ca²⁺ release through ryanodine receptors (Fraysse et al., 2003). As shown in Fig. 7A, caffeine (50 mM) led to small Ca²⁺ transients, as previously reported for fast-twitch skeletal muscle fibers (Fraysse et al., 2003). Peak Ca²⁺ responses were not significantly different between C57BL/6J and mdx^5cv fibers (220.3±20 nM and 196.6±30.8 nM in C57BL/6J and mdx^5cv fibers; respectively, Fig. 7A). Although peak Ca²⁺ responses were identical in both types of fibers, caffeine-induced Mn²⁺ entry was strongly increased in mdx^5cv fibers, in accordance with previous results (28.1±2.3% and 51.1±3.9% for C57BL/6J and mdx^5cv fibers, respectively; Fig. 7B,C). When both types of fibers were incubated with BTP2 (5 µM), Mn²⁺ entry triggered by caffeine was nearly completely blocked (Fig. 7B,C). Similar results were obtained with the SOC blocker Gd³⁺ (not shown), indicating that caffeine triggered Mn²⁺ entry through SOC. Pretreatment of mdx^5cv fibers with BEL (5 µM) strongly decreased caffeine-induced Mn²⁺ entry (51.1±3.9% to 14.3±1.9% for control and BEL treated mdx^5cv fibers). In accordance with previous results, iPLA₂ inhibition had a much weaker effect in C57BL/6J fibers (28.1±2.3% to 18.9±1.3% for control and BEL treated C57BL/6J fibers; Fig. 7B,C). These results indicate that whatever the extent of Ca²⁺-store depletion or the mechanism responsible for store discharge (SERCA inhibition by thapsigargin or Ca²⁺ release through ryanodine receptors), iPLA₂ is involved in the regulation of SOC in mdx^5cv fibers.

Effect of melittin on Mn²⁺ entry in mdx^5cv fibers
To further investigate the involvement of iPLA₂ in the regulation of store-operated Ca²⁺ entry in mdx^5cv fibers, we tested the effect of melittin on Mn²⁺ entry assessed by the quench of Fura-2 fluorescence. Melittin, a toxin from bee venom, has been demonstrated to activate PLA₂ and subsequent release of arachidonic acid and lysophospholipids in various types of cells (Choi et al., 1992; Eintracht et al., 1998; Sharma, 1993). As illustrated in Fig. 8A, a 2 second application of melittin (5 µM) on mdx^5cv fibers led to Mn²⁺ entry. Incubation of the cells with the SOC blocker BTP2 (5 µM) strongly reduced melittin-induced Mn²⁺ entry (from 39.2±3.3% to 10.6±2.1%; Fig. 8A,B), suggesting that melittin stimulates Mn²⁺ influx through SOC. Preincubation of fibers with the iPLA₂ inhibitor BEL also reduced melittin-induced Mn²⁺ entry (from 39.2±3.3% to 15.6±4.8%; Fig. 8A,B). These results suggest that iPLA₂ is activated by melittin, in turn triggering Mn²⁺ entry through SOC.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Effect of BTP2 and BEL on Mn2+ entry triggered by the PLA2 activator melittin. (A) Melittin (5 µM) was applied for 2 seconds onto isolated mdx5cv fibers and Mn2+ entry was measured by the quench of Fura-2 fluorescence recorded at an excitation wavelength of 360 nm. Prior to melittin application, fibers were incubated with a solution containing 0.5 mM MnCl2 and 1.7 mM CaCl2. On the same graph are shown results from a control experiment and from experiments performed on fibers preincubated with either BTP2 (5 µM) for 10 minutes or BEL (10 µM) for 20 minutes (hatched trace). (B) Compiled data showing the percentage of fluorescence decrease 90 seconds after melittin application in control condition and when fibers had been preincubated with either BTP2 or BEL. Vertical hatched line on recordings shown in A indicates the time for measurements of fluorescence decrease. Numbers of fibers tested (from 2 mdx5cv mice) are indicated on bar graphs. (C) Top: western blot of iPLA2 performed on FDB and EDL (extensor digitorum longus) C57BL/6J and mdx5cv muscles. Bottom: quantitative analysis of iPLA2 amounts in FDB and EDL muscles from C57BL/6J and mdx5cv mice. Numbers of mice used are indicated on bar graphs. (D) Left panel: western blot of SERCA1 performed on C57BL/6J and mdx5cv FDB muscles. Right panel: quantitative analysis of SERCA1 amounts in FDB muscles from C57BL/6J and mdx5cv mice. Numbers of mice used are indicated on bar graphs.

Fig. 8D shows that the anti-SERCA1 antibody recognized a specific band of around 110 kDa for C57BL/6J and mdx^5cv FDB muscles, corresponding to the molecular mass of SERCA1, the major SERCA isoform in fast twitch muscle (Rossi and Dirksen, 2006). Quantitative analysis revealed that the expression of SERCA1 is essentially the same in FDB muscles from C57BL/6J and mdx^5cv mice (Fig. 8D).

	Discussion

Top Summary Introduction Results Discussion Materials and Methods References

 
We report here on our investigations regarding regulation of store-operated Ca²⁺ influx in dystrophic (mdx^5cv) and normal (C57BL/6J) skeletal muscle fibers using cytosolic Ca²⁺ measurements and the manganese quench method.

In skeletal muscle, Ca²⁺ transients triggered by nicotinic receptor activation or KCl-induced depolarization are due to Ca²⁺ release from the SR through ryanodine receptors, which are directly activated by L-type voltage-gated Ca²⁺ channels upon plasma membrane depolarization (Berchtold et al., 2000). Thus, L-type voltage-gated Ca²⁺ channels act as voltage-sensors and Ca²⁺ influx through these channels is not needed for Ca²⁺ release through ryanodine receptors (Lamb, 2002; O'Brien et al., 2002). Our results clearly show that the amplitude of Ca²⁺ transients triggered by KCl depolarization is not reduced in Ca²⁺-free solution in both normal and dystrophic fibers, indicating that FDB fibers used in this study have a characteristic skeletal-type excitation-contraction coupling mechanism.

Basal cytosolic Ca²⁺ concentrations were very similar in normal and dystrophic FDB fibers, in agreement with previous reports (Collet et al., 1999; De Backer et al., 2002; Tutdibi et al., 1999; Vandebrouck et al., 2002). Peak values of cytosolic Ca²⁺ transients triggered by KCl depolarization were also not significantly different between C57BL/6J and mdx^5cv fibers in both normal and Ca²⁺-free solution, suggesting that mechanisms involved in Ca²⁺ release are not altered in dystrophic fibers, as previously reported for electrically-evoked Ca²⁺ transients (Collet et al., 1999; Gillis, 1996; Plant and Lynch, 2003; Tutdibi et al., 1999).

As opposed to the amplitudes of KCl-induced Ca²⁺ transients, the kinetic properties of these were altered in dystrophic fibers, which exhibited a slower decay phase than normal ones. Similar findings have been reported for electrically evoked Ca²⁺ transients in mdx FDB fibers, ACh-evoked Ca²⁺ transients in dystrophic myotubes or relaxation speeds after electrical stimulation of dystrophic myotubes (Nicolas-Metral et al., 2001; Rivet-Bastide et al., 1993; Tutdibi et al., 1999; Woods et al., 2004).

A slower decay phase of Ca²⁺ transients in mdx^5cv fibers may be due to lowered SERCA activity, to increased Ca²⁺ influx, to increased intracellular Ca²⁺ release or a combination of these effects. When experiments were performed in Ca²⁺-free solution, to prevent Ca²⁺ influx, half times of decay were significantly reduced in both types of fibers, indicating that Ca²⁺ entry is primarily responsible for the slow decay phase of these responses, although one can not exclude a minor involvement of intracellular Ca²⁺ release. Interestingly, in Ca²⁺-free solution, the half-time of decay of Ca²⁺ transients was similar for both types of fibers, suggesting that Ca²⁺ removal capabilities, mostly due to SERCA function, are not altered in mdx^5cv fibers. In accordance with these findings, SERCA1, the major SERCA isoform in fast-twitch muscle (Rossi and Dirksen, 2006), was found to be similarly expressed in C57BL/6J and mdx^5cv FDB muscles. Therefore Ca²⁺ influx appears to be mainly responsible for the slow decay of KCl-induced Ca²⁺ transients in both types of fibers, raising the question about the Ca²⁺ entry pathway.

It is unlikely that Ca²⁺ entry occurred through L-type voltage-gated Ca²⁺ channels, because nifedipine, known to block Ca²⁺ currents through L-type voltage gated Ca²⁺ channels in skeletal muscle fibers, was used throughout our investigations (O'Connell and Dirksen, 2000). However, the SOC blocker BTP2 reduced the half-time of decay of Ca²⁺ transients in both types of fibers, suggesting that the slow decay could be due to opening of SOC. This is consistent with reports showing activation of store-operated Ca²⁺ entry when SR calcium stores were depleted in skeletal muscle cells (Kurebayashi and Ogawa, 2001; Ma and Pan, 2003). Slower decay phase in mdx^5cv fibers could then be due to enhanced store-operated Ca²⁺ entry during Ca²⁺ responses.

This hypothesis was tested by experiments using thapsigargin and caffeine. Indeed, when SR Ca²⁺ stores were depleted with thapsigargin or caffeine, a greatly enhanced Ca²⁺/Mn²⁺ entry through SOC was found in dystrophic fibers. These results point to an increased activity of SOC following Ca²⁺-store depletion in dystrophic FDB fibers, in accordance with other reports (Vandebrouck et al., 2006; Vandebrouck et al., 2002).

What could be the trigger for store-dependent Ca²⁺ entry and why is the decay of Ca²⁺ transients slower in dystrophic fibers? Our results suggest that iPLA₂ is of central importance in regulating SOC in dystrophic fibers. We found that exposure of fibers with iPLA₂ inhibitors normalized the decay of Ca²⁺ transients in mdx^5cv fibers without affecting transients of C57BL/6J fibers. Experiments using thapsigargin or caffeine to deplete SR Ca²⁺ stores, showed that the iPLA₂ inhibitor BEL greatly reduced store-operated Ca²⁺/Mn²⁺ entry in mdx^5cv fibers but had only a weak effect in C57BL/6J fibers. Altogether, these results indicate that iPLA₂ activity may be the cause of enhanced store-operated Ca²⁺ entry in mdx^5cv fibers, and therefore that the activity of SOC must be different between C57BL/6J and mdx^5cv fibers.

The involvement of iPLA₂ in the regulation of SOC in dystrophic fibers is further supported by the fact that melittin, a toxin from bee venom and potent PLA₂ activator (Choi et al., 1992; Eintracht et al., 1998; Sharma, 1993), triggered Mn²⁺ entry through SOC, which was blocked when iPLA₂ was inhibited by BEL. The blockade of Mn²⁺ entry by both the SOC blocker BTP2 and the iPLA₂ inhibitor BEL indicates that melittin triggers hyperactivation of iPLA₂, resulting in SOC opening.

Various PLA₂ isoforms, including iPLA₂, have been shown to regulate store-operated Ca²⁺ entry in other types of cells (Hichami et al., 2002; Martinez and Moreno, 2005; Rzigalinski et al., 1999; Smani et al., 2004; Smani et al., 2003; Zablocki et al., 2000). Enhanced store-operated Ca²⁺ entry in mdx^5cv fibers could be explained by higher expression levels of iPLA₂ in dystrophic muscles compared with normal ones. Interestingly, a greatly increased total PLA₂ activity has been found in muscles from DMD patients (Lindahl et al., 1995). Since numerous PLA₂ products have been shown to be activators of cationic/SOC channels (Martinez and Moreno, 2005; Rzigalinski et al., 1999; Smani et al., 2004; So et al., 2005; Terasawa et al., 2002), enhanced production of the hydrolysis products of PLA₂ could be responsible for the enhanced Ca²⁺ entry observed in mdx^5cv fibers, as proposed in Fig. 9.

View larger version (28K): [in this window] [in a new window]   Fig. 9. Hypothetical model for enhanced store-operated Ca2+ entry in dystrophic skeletal muscle fibers. In skeletal muscle, ryanodine receptors (RyR) are allosterically activated by L-type voltage-gated Ca2+ channels (VGCC) when plasma membrane (PM) is depolarized (V). SR Ca2+ released through ryanodine receptors triggers contraction and Ca2+ is pumped back into the SR by the SERCA. Ca2+-store depletion occurring during Ca2+ release through ryanodine receptors or when SERCA is blocked by thapsigargin triggers iPLA2 activation, possibly by the release of calcium influx factor (CIF). Hydrolysis products of iPLA2 (most likely lysophospholipids) may be responsible for SOC stimulation and Ca2+ influx. Overexpression of iPLA2 in dystrophic muscle is likely to be responsible for enhanced Ca2+ influx through SOC.

	Materials and Methods

Top Summary Introduction Results Discussion Materials and Methods References

 
Cell preparation
Normal (C57BL/6J) or dystrophic (mdx^5cv) mice (3-4 months old) were killed by cervical dislocation. Flexor digitorum brevis (FDB) muscles from both legs were removed quickly, and incubated in Dulbecco's Modified Eagle's Medium (DMEM) containing 1 mg/ml collagenase type IA (Sigma) at 37°C for 60 minutes. Tissues were placed in enzyme-free DMEM and gently triturated using fire-polished Pasteur pipettes to release fibers. Cells were stored on glass coverslips coated with Matrigel (400 µg/ml, Collaborative Research) and maintained in short-term primary culture in DMEM containing 10% fetal calf serum and 0.5% ciproxine (Bayer). Fibers were kept in an incubator gassed with 95% air-5% CO₂ at 37°C. Cells were used from 18-28 hours after isolation.

Calcium imaging
Intracellular Ca²⁺ concentration was monitored with the fluorescent Ca²⁺ indicator Fura-2AM (acetoxymethylester form of Fura-2, cell permeant). Before loading, cells were washed with physiological salt solution (PSS), consisting of (in mM): 130 NaCl, 5.6 KCl, 1 MgCl₂, 1.7 CaCl₂, 11 glucose, and 10 Hepes, pH 7.4. Cells were incubated for 30 minutes in PSS containing 1 µM Fura-2AM, washed to remove extracellular Fura-2, and allowed to de-esterify for 20 minutes. To minimize potential compartmentalization of the dye, Fura-2 loading was performed at room temperature.

Ca²⁺ measurements were performed using an inverted microscope (Zeiss Axiovert 200M) with a x20 objective, and images were acquired with an intensified CCD camera (Extended Isis, Photonic Science). The fiber culture chamber was placed on the stage of the microscope. Ultraviolet light, emitted from a 75 W xenon lamp (Visitron Systems), passed through a high-speed monochromator (Visitron Systems), which selects alternately excitation wavelengths of 340 and 380 nm. Fluorescence emitted by cells was band-passed filtered at around 510 nm and collected by the CCD camera. Metafluor software (Universal Imaging Corporation) was used to record and analyze fluorescence measurements. Fluorescence was processed by correcting each image for background fluorescence and calculating 340/380 nm fluorescence ratios on a pixel-by-pixel basis. [Ca²⁺]_i was calculated according to the following equation (Grynkiewicz et al., 1985):

[Ca²⁺]_i=K_dx(F_380max/F_380min)x(R-R_min)/(R_max-R) where K_d is the dissociation constant for the Fura-2/Ca²⁺ complex (135 nM at 20°C) (Grynkiewicz et al., 1985), R_min and R_max are the fluorescence ratios of Fura-2 in the absence of and in the presence of saturating [Ca²⁺], respectively, and R is the experimental ratio. F_380max and F_380min are the Fura-2 fluorescence intensities at 380 nm in the absence and in the presence of a saturating [Ca²⁺], respectively. In situ calibration of Fura-2 was carried out in both types of fibers. R_min was determined by bathing the cells in a Ca²⁺-free solution containing 5 mM EGTA, followed by permeabilization with saponin (100 µg/ml). R_max was determined by bathing fibers in a 2 mM Ca²⁺ containing solution, followed by permeabilization with saponin (100 µg/ml). In both cases, fibers were previously incubated for 10 minutes with BTS (50 µM), to prevent contraction (Cheung et al., 2002; Woods et al., 2004). R_min and R_max were not found to be different between C57BL/6J and mdx^5cv FDB fibers. Ca²⁺ transients were measured in the whole perimeter of fibers. All experiments were carried out in PSS at room temperature (22°C).

Manganese influx measurements
The manganese quench technique was used to estimate divalent cation influx through the sarcolemma (Kurebayashi and Ogawa, 2001; Tutdibi et al., 1999). Fibers were first loaded with Fura-2 as previously described and MnCl₂ (0.5 or 1 mM) was added to the bath solution. As Mn²⁺ quenches Fura-2 fluorescence, Mn²⁺ influx through the sarcolemma triggers a decrease of the fluorescence of Fura-2 loaded cells. To measure Mn²⁺ influx, cells were excited at 360 nm (isobestic point of Fura-2). As Mn²⁺ has a similar permeability as Ca²⁺ through most plasma membrane Ca²⁺ channels, the quench of Fura-2 fluorescence when Fura-2 is excited at 360 nm allows estimation of Mn²⁺ entry through plasma membrane Ca²⁺ channels (Tutdibi et al., 1999). Mn²⁺ influx was estimated by the percentage decrease of Fura-2 fluorescence 90 seconds after stimulation was applied to fibers.

Drug application and solutions
PSS containing test compounds or high-KCl solutions were quickly applied to single cells by pressure ejection using a pinch valve pressurized perfusion system (ALA Scientific Instruments, USA) connected to a quartz micromanifold, with an output tip size of 100 µm. The micromanifold was mounted on a Leitz micromanipulator, to stimulate individual skeletal muscle fibers for the period indicated on the records. Stock solutions of compounds applied on cells were diluted in PSS. Normal High-KCl solution contained (in mM): 25.6 NaCl, 110 KCl, 1 MgCl₂, 11 glucose, 10 Hepes, and 1.7 CaCl₂ (pH 7.4). A Ca²⁺-free/high-KCl solution was used in some experiments. Nifedipine (1 µM) was present in High-KCl and PSS solutions containing compounds that were applied to fibers by pressure ejection with the perfusion system.

Immunoblotting
FDB and EDL (extensor digitorum longus) muscle proteins from mdx^5cv and C57BL/6J mice were extracted in Laemmli buffer containing dithiothreitol (100 mM). Extracts were incubated for 30 minutes at 4°C and 30 minutes at room temperature followed by 5 minutes at 95°C. Extracts were centrifuged at 10,000 g and protein determination was performed. For western blot analysis, FDB extracts isolated from C57BL/6J and mdx^5cv mice were loaded (30 µg protein/well) and separated on 10% SDS-PAGE minigels. Proteins were then transferred to nitrocellulose membranes for 90 minutes at 100 volt in a transfer buffer (192 mM glycine, 25 mM Tris.Base and 10% methanol). Membranes were incubated overnight in blocking buffer [20 mM Tris.Base, 500 mM NaCl, 0.1% Tween 20 and 5% non fat dried milk (pH 7.4)], and then incubated 3 hours with the primary antibody (rabbit anti-iPLA₂ antibody, Cayman, USA) at a 1/1000 dilution in TBST. After extensive washing, membranes were incubated for 1 hour with the secondary antibody diluted 1/10,000 (anti-rabbit, Amersham). Specific antigen detection was performed using an ECL kit (Amersham). Quantitative analysis of iPLA₂ specific bands was performed by normalization with Ponceau S using Image J software (NIH Image). Western blot analysis of SERCA1 expression was performed using mouse anti-SERCA1 antibody (Affinity Bioreagents) at 1/1000 and anti-mouse antibody (Amersham) at 1/50,000.

Chemicals
Arachidonyltrifluoromethyl ketone (AACOCF₃), [N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide] (BTP2), N-benzyl-p-toluene-sulfonamide (BTS) and thapsigargin were from Calbiochem. Acetylcholine, caffeine, collagenase type IA and bromoenol lactone (BEL) were from Sigma. Fura-2AM was from Molecular Probes. Ethylene glycol-bis(2-aminoethyl)-N,N,N',N'-tetra acetic acid (EGTA) was from Fluka.

Statistics
Results are expressed as means ± s.e.m. Significance was tested by means of Student's t-test and P values of <0.05 were considered as significant.

	Acknowledgments

 
This work was supported by grants from the Swiss National Science Foundation (31-68315.02 and 31-109981.05), the Swiss Foundation for Research on Muscular Diseases, the `Association Française contre les Myopathies' and the Leenaards Foundation. We thank Drs B. Gähwiler and U. Schibler for proofreading the manuscript.

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