Christian Cognard

Regulation of capacitative calcium entries by α1-syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of α1-syntrophin

Calcium mishandling in Duchenne dystrophic muscle suggested that dystrophin, a membrane‐associated cytoskeleton protein, might regulate calcium signaling cascade such as calcium influx pathway. It was previously shown that abnormal calcium entries involve uncontrolled stretch‐activated currents and store‐operated Ca2+ currents supported by TRPC1 channels. Moreover, our recent work demonstrated that reintroduction of minidystrophin in dystrophic myotubes restores normal capacitative calcium entries (CCEs). However, until now, no molecular link between the dystrophin complex and calcium entry channels has been described. This study is the first to show by coimmunoprecipitation assays the molecular association of TRPC1 with dystrophin and α1‐syntrophin in muscle cells. TRPC1 was also associated with α1‐syntrophin in dystrophic muscle cells independently of dystrophin. Furthermore, glutathione S‐transferase (GST) pull‐down assays showed that TRPC1 binds to the α1‐syntrophin PDZ domain. Transfected recombinant α1‐syntrophin formed a complex with TRPC1 channels and restored normal CCEs in dystrophic muscle cells. We suggest that normal regulation of CCEs in skeletal muscle depends on the association between TRPC1 channels and α1‐syntrophin that may anchor the store‐operated channels to the dystrophin‐associated protein complex (DAPC). The loss of this molecular association could participate in the calcium alterations observed in dystrophic muscle cells. This study provides a new model for the regulation of calcium influx by interaction with the scaffold of the DAPC in muscle cells.—Vandebrouck, A., Sabourin, J., Rivet, J., Balghi, H., Sebille, S., Kitzis, A., Raymond, G., Cognard, C., Bourmeyster, N., Constantin, B. Regulation of capacitative calcium entries by α1‐syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of α1‐syntrophin. FASEB J. 21, 608–617 (2007)

Abnormal calcium homeostasis in Duchenne muscular dystrophy myotubes contracting in vitro

Resting intracellular calcium activity was recorded in three kinds of human muscle cells in culture: normal (control) and dystrophic (DMD and FSH), by means of a ratiometric fluorescence method using the calcium probe Indo-1 under laser illumination. DMD cells are characterized by a lack of dystrophin whereas FSH cells express normal dystrophin. The aim of this study was to determine whether, in dystrophin-deficient muscle cells (DMD), contraction destabilized internal calcium homeostasis. Muscle cells were cocultured with rat spinal cord explants to improve the maturation of human myotubes up to the stage where contraction appears. The resting intracellular calcium level was significantly higher in contracting DMD cells (107 +/- 8 nM; n = 44) compared to control cells (66 +/- 6 nM; n = 43) or in FSH cells (56 +/- 6 nM; n = 35). DMD myotubes cocultured in the presence of TTX which inhibited contractile activity, did not develop an increase in free cytosolic Ca2+ concentration. The amplitudes of calcium transients elicited by exposure to acetylcholine (ACh) or high K+ medium (100K) were significantly higher in contracting DMD myotubes than in control ones. The extra-responses were not observed in DMD myotubes cocultured with TTX. This study strongly suggest that: (i) contraction is a dominant factor contributing to Ca2+ abnormalities in DMD cells; and (ii) contracting dystrophin-deficient cells have defective calcium handling mechanisms during electrical events which involve sarcolemma.

Regulation of TRPC1 and TRPC4 Cation Channels Requires an α1-Syntrophin-dependent Complex in Skeletal Mouse Myotubes

The dystrophin-associated protein complex (DAPC) is essential for skeletal muscle, and the lack of dystrophin in Duchenne muscular dystrophy results in a reduction of DAPC components such as syntrophins and in fiber necrosis. By anchoring various molecules, the syntrophins may confer a role in cell signaling to the DAPC. Calcium disorders and abnormally elevated cation influx in dystrophic muscle cells have suggested that the DAPC regulates some sarcolemmal cationic channels. We demonstrated previously that mini-dystrophin and α1-syntrophin restore normal cation entry in dystrophin-deficient myotubes and that sarcolemmal TRPC1 channels associate with dystrophin and the bound PDZ domain of α1-syntrophin. This study shows that small interfering RNA (siRNA) silencing of α1-syntrophin dysregulated cation influx in myotubes. Moreover, deletion of the PDZ-containing domain prevented restoration of normal cation entry by α1-syntrophin transfection in dystrophin-deficient myotubes. TRPC1 and TRPC4 channels are expressed at the sarcolemma of muscle cells; forced expression or siRNA silencing showed that cation influx regulated by α1-syntrophin is supported by TRPC1 and TRPC4. A molecular association was found between TRPC1 and TRPC4 channels and the α1-syntrophin-dystrophin complex. TRPC1 and TRPC4 channels may form sarcolemmal channels anchored to the DAPC, and α1-syntrophin is necessary to maintain the normal regulation of TRPC-supported cation entry in skeletal muscle. Cation channels with DAPC form a signaling complex that modulates cation entry and may be crucial for normal calcium homeostasis in skeletal muscles.

Myoblast fusion requires cytosolic calcium elevation but not activation of voltage-dependent calcium channels

Many studies of in vitro skeletal myogenesis have demonstrated that fusion of myoblasts into multinucleated myotubes is regulated by calcium-dependent processes. Calcium ions appear to be necessary at the outer face of the membrane, and an additional internal calcium increase seems required to promote fusion of aligned myoblasts. It has been proposed that a calcium influx could take place prior to fusion and that this may be mediated by voltage-dependent calcium channels. Previously, we showed that two types of voltage-dependent calcium currents were expressed in multinucleated myotubes but not in rat myoblasts growing in primary culture before the withdrawal of the growth medium. We also showed that the previous formation of multinucleated synticia was not a prerequisite of developmental appearance of calcium currents, suggesting that the two events were time-correlated but not sequentially dependent. These features led us to investigate changes in internal calcium activity and the possible appearance of voltage-dependent calcium influx pathways just after the promotion of fusion by the change of culture medium. The results confirm that a rise in cytosolic calcium activity occurs slightly before fusion in confluent myoblasts and remained in newly formed myotubes. Reducing this elevation by internal calcium buffering lowered myoblast fusion and, reciprocally, blocking cell fusion prevented calcium increase. Treatment with the organic calcium channel blockers nifedipine (5 microM) and PN 200-110 (1 microM) did not alter cytosolic calcium changes nor cell fusion, and voltage-dependent calcium currents were never observed by the perforated patch-clamp technique in aligned fusion-competent myoblasts. Other voltage-operated mechanisms of calcium rise were not detected since depolarization with hyperpotassium solutions failed to elicit increases in intracellular calcium. On the contrary, acetylcholine was able to promote extracellular calcium-dependent calcium transients. Our results confirm the requirement of an increase in resting calcium during fusion, but do not support the hypothesis of an influx through voltage-dependent channels or other voltage-operated pathways. The elevation of internal calcium activity may result from other mechanisms, such as a cholinergic action for example.

New insights in the regulation of calcium transfers by muscle dystrophin-based cytoskeleton: implications in DMD

Calcium mishandling in Duchenne muscular dystrophy (DMD) suggested that dystrophin, a membrane-associated cytoskeleton protein, may regulate calcium-signalling cascades such as calcium entries. Calcium overload in human DMD myotubes is dependent on their contractile activity suggesting the involvement of channels being activated during contraction and/or calcium release. Forced expression of mini-dystrophin in dystrophin-deficient myotubes, reactivates appropriate sarcolemmal expression of dystrophin-associated proteins and restores normal calcium handling in the cytosol. Furthermore, the recombinant mini-dystrophin reduced the store-operated calcium influx across the sarcolemma, and the mitochondrial calcium uptake during this influx. A slow component of calcium release dependent on IP3R, as well as the production of IP3, were also reduced to normal levels by expression of mini-dystrophin. Our studies provide a new model for the convergent regulation of transmembrane calcium influx and IP3-dependent calcium release by the dystrophin-based cytoskeleton (DBC). We also suggest molecular association of such channels with DBC which may provide the scaffold for assembling a multiprotein-signalling complex that modulates the channel activity. This suggests that the loss of this molecular association could participate in the alteration of calcium homeostasis observed in DMD muscle cells.

Intracellular calcium transients induced by different kinds of stimulus during myogenesis of rat skeletal muscle cells studied by laser cytofluorimetry with Indo-1

Resting intracellular calcium levels and intracellular calcium transients induced by three types of stimulus (acetylcholine, high potassium and caffeine) were recorded, during in vitro myogenesis, by means of a ratiometric fluorescence method using the calcium probe Indo-1 under laser illumination. Resting levels seemed to decrease with the age of cultured cells and the depolarization-induced transients, through 100 mM K+ or Ach application, were progressively faster and larger as the muscle cells developed. An additive mechanism, likely due to calcium entry into the cell through nicotinic acetylcholine receptors, could explain the differences observed in Ach-induced responses as compared with the 100 mM K(+)-induced ones. In myoballs (the older cells) the calcium transients exhibited progressively a biphasic shape. From data obtained in different conditions (tetrodotoxin, nifedipine, strontium and free Ca EGTA) and those indicating the appearance of caffeine-releasable intracellular calcium stores only at 2-3 days stage, and from the previously reported developmental appearance of calcium currents and contraction, it was proposed that, in young myotubes, the calcium transients were more dependent on extracellular calcium than in older cells. These developmental data are discussed in the light of a known model of the in situ biogenesis of the structures involved in excitation-contraction coupling (ECC) like transverse tubules and triads.

Bcl-2 overexpression prevents calcium overload and subsequent apoptosis in dystrophic myotubes

Duchenne muscular dystrophy (DMD) is a lethal disease caused by the lack of the cytoskeletal protein dystrophin. Altered calcium homoeostasis and increased calcium concentrations in dystrophic fibres may be responsible for the degeneration of muscle occurring in DMD. In the present study, we used subsarcolemmal- and mitochondrial-targeted aequorin to study the effect of the antiapoptotic Bcl-2 protein overexpression on carbachol-induced near-plasma membrane and mitochondrial calcium responses in myotubes derived from control C57 and dystrophic (mdx) mice. We show that Bcl-2 overexpression decreases subsarcolemmal and mitochondrial calcium overload that occurs during activation of nicotinic acetylcholine receptors in dystrophic myotubes. Moreover, our results suggest that overexpressed Bcl-2 protein may prevent near-plasma membrane and mitochondrial calcium overload by inhibiting IP3Rs (inositol 1,4,5-trisphosphate receptors), which we have shown previously to be involved in abnormal calcium homoeostasis in dystrophic myotubes. Most likely as a consequence, the inhibition of IP3R function by Bcl-2 also inhibits calcium-dependent apoptosis in these cells.

Hypoosmotic shocks induce elevation of resting calcium level in duchenne muscular dystrophy myotubes contracting in vitro

In Duchenne muscular dystrophy (DMD) muscle cells which lack dystrophin, contraction seems to be a dominant factor contributing to the abnormal elevated intracellular calcium level. Human normal and DMD contracting myotubes cocultured with nervous cells were exposed to a hypotonic medium to mimic contraction-induced mechanical stress on the membrane, and the cytoplasmic calcium activity was simultaneously monitored (Indo-1). Hypotonic shocks induced a reversible [Ca2+]i increase in 81% of the DMD cells vs. 54% of control. In addition, responses were qualitatively different: most of DMD myotubes displayed a fast increase of Ca2+ flowing from the edge of the myotube while the response in normal cells was slow and diffuse. The fact that these responses were not affected by ryanodine, was in favour of an external source of Ca2+ involved in the hypoosmotic shocks. The localized increase of Ca2+ in DMD myotubes, inhibited by Gd3+, could result from sites of high mechanosensitive channel activity or density which could constitute a pathway for Ca2+ entry provided these cells contract.

Effects of two medicinal plants Psidium guajava L. (Myrtaceae) and Diospyros mespiliformis L. (Ebenaceae) leaf extracts on rat skeletal muscle cells in primary culture

Crude decoction, aqueous and ethanolic extracts of two medicinal plants (Psidium guajava and Diospyros mespiliformis), widely used in the central plateau of Burkina Faso to treat many diseases were evaluated for their antagonistic effects on caffeine induced calcium release from sarcoplasmic reticulum of rat skeletal muscle cells. These different extracts showed a decrease of caffeine induced calcium release in a dose dependent manner. Comparison of the results showed that Psidium guajava leaf extracts are more active than extracts of Diospyros mespiliformis and that crude decoctions show better inhibitory activity. The observed results could explaine their use as antihypertensive and antidiarrhoeal agents in traditional medicine, by inhibiting intracellular calcium release.

Changes in cytosolic resting ionized calcium level and in calcium transients during in vitro development of normal and Duchenne muscular dystrophy cultured skeletal muscle measured by laser cytofluorimetry using indo-1.

Intracellular calcium activity was recorded during in vitro myogenesis of human normal and DMD muscle, using the calcium probe Indo-1 under laser illumination, at rest and during different kinds of stimulation (acetylcholine, high K+, caffeine). In myoblasts, the resting intracellular calcium level was significantly larger in DMD cells (89 +/- 9 nM; n = 40 vs 37 +/- 5 nM; n = 22) but there was no significant difference in myotubes, after fusion (44 +/- 4 nM; n = 34 vs 36 +/- 4 nM; n = 52). A similar evolution was observed in cells cultured from FSH biopsies. The amplitude of ACh- and high K(+)-induced calcium transients was significantly halved in DMD myotubes as compared to control ones and non-significantly decreased for caffeine responses. Some alterations in the kinetics of responses were observed in DMD muscle: the rising phases of ACh- and high K(+)-elicited transients and the decaying phase of the ACh-responses were significantly slowed down. It is concluded that: (i) in aneurally cultured human muscle, an increase in the basal level of internal calcium can occur at early stages of myogenesis before the expression of the dystrophin gene; and (ii) the changes in calcium transients induced by depolarization or direct stimulation of sarcoplasmic reticulum are not susceptible of inducing a calcium overload in DMD cells.