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

Journal of Cell Science 119, 3733-3742 (2006)
Published by The Company of Biologists 2006
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Ca2+-independent phospholipase A2 enhances store-operated Ca2+ entry in dystrophic skeletal muscle fibers

François-Xavier Boittin, Olivier Petermann, Carole Hirn, Peggy Mittaud, Olivier M. Dorchies, Emmanuelle Roulet and Urs T. Ruegg*

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


Figure 1
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  		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.

 

Figure 2
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  		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.

 

Figure 3
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  		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.

 

Figure 4
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  		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.

 

Figure 5
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  		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.

 

Figure 6
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  		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.

 

Figure 7
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  		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.

 

Figure 8
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  		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.

 

Figure 9
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  		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 ({Delta}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.

 

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© The Company of Biologists Ltd 2006