Bruno Allard

T-tubule disorganization and defective excitation-contraction coupling in muscle fibers lacking myotubularin lipid phosphatase

Skeletal muscle contraction is triggered by the excitation-contraction (E-C) coupling machinery residing at the triad, a membrane structure formed by the juxtaposition of T-tubules and sarcoplasmic reticulum (SR) cisternae. The formation and maintenance of this structure is key for muscle function but is not well characterized. We have investigated the mechanisms leading to X-linked myotubular myopathy (XLMTM), a severe congenital disorder due to loss of function mutations in the MTM1 gene, encoding myotubularin, a phosphoinositide phosphatase thought to have a role in plasma membrane homeostasis and endocytosis. Using a mouse model of the disease, we report that Mtm1-deficient muscle fibers have a decreased number of triads and abnormal longitudinally oriented T-tubules. In addition, SR Ca2+ release elicited by voltage-clamp depolarizations is strongly depressed in myotubularin-deficient muscle fibers, with myoplasmic Ca2+ removal and SR Ca2+ content essentially unaffected. At the molecular level, Mtm1-deficient myofibers exhibit a 3-fold reduction in type 1 ryanodine receptor (RyR1) protein level. These data reveal a critical role of myotubularin in the proper organization and function of the E-C coupling machinery and strongly suggest that defective RyR1-mediated SR Ca2+ release is responsible for the failure of muscle function in myotubular myopathy.

Intracellular calcium signals measured with indo‐1 in isolated skeletal muscle fibres from control and mdx mice

1 Intracellular free calcium concentration ([Ca2+]i) was measured with the fluorescent indicator indo‐1 in single skeletal fibres enzymatically isolated from the flexor digitorum brevis and interosseus muscles of control and dystrophic mdx C57BL/10 mice. Measurements were taken from a portion of fibre that was voltage clamped to allow detection of depolarization‐induced changes in [Ca2+]i. 2 The mean (±s.e.m.) initial resting [Ca2+]i from all control and mdx fibres tested was 56 ± 5 nm (n= 72) and 48 ± 7 nm (n= 57), respectively, indicating no significant overall difference between the two groups. However, when comparing a batch of control and mdx fibres obtained from mice older than ∼35 weeks, resting [Ca2+]i was significantly lower in mdx (16 ± 4 nm, n= 11) than in control fibres (71 ± 10 nm, n= 14). 3 Changes in [Ca2+]i elicited by short (5–35 ms) depolarizing pulses from −80 to 0 mV showed similar properties in control and mdx fibres. After a 5 ms duration pulse the mean time constant of [Ca2+]i decay was, however, significantly elevated in mdx as compared to control fibres, by a factor of 1.5–2. For longer pulses, no significant difference could be detected. 4 In response to 50 ms duration depolarizing pulses of various amplitudes the threshold for detection of an [Ca2+]i change and the peak [Ca2+]i reached for a given potential were similar in control and mdx fibres. 5 Overall results show that mdx skeletal muscle fibres are quite capable of handling [Ca2+]i at rest and in response to membrane depolarizations.

Pharmacological properties of ATP-sensitive K+ channels in mammalian skeletal muscle cells

The patch-clamp technique (single-channel recordings) was used to study the effects of glibenclamide and some channel openers on the KATP channel in mouse skeletal muscle. In outside/out membrane patches, glibenclamide reversibly inhibited KATP channel activity in a dose-dependent manner with an apparent Ki of 190 nM. In inside/out membrane patches, RP 61419 increased KATP channel activity both in the absence and in the presence of internal ATP while other K+ channel openers such as nicorandil and cromakalim required the presence of internal ATP to evoke channel activation. The half-maximal activity effect for cromakalim, with 0.5 mM ATP at the cytoplasmic face, was observed at about 220 microM. Pinacidil was unable to activate the KATP channel in the absence of internal ATP and could even reduce channel opening in situations where activity was high in the control. In the presence of internal Mg2+, activation by pinacidil occurred when ATP or low and weakly activating concentrations of ADP were present at the cytoplasmic side. Pinacidil activation could also be observed in the presence of ATP or ADP when Mg2+ was absent from the internal solution. The mechanism of action of pinacidil is discussed in terms of interactions between the different nucleotide regulatory sites and the K+ channel opener binding site of the KATP channel. Half-maximum activation of the KATP channel in the presence of 0.5 mM ATP at the cytoplasmic face was observed at 125 microM pinacidil.

Nucleotide diphosphates activate the ATP-sensitive potassium channel in mouse skeletal muscle

Patch-clamp techniques were used to study the effects of internal nucleotide diphosphates on the KATP channel in mouse skeletal muscle. In inside-out patches, application of GDP (100 μM) and ADP (100 μM) reversibly increased the channel activity. In the presence of internal Mg2+ (1 mM), low concentrations of ADP (<300 μM) enhanced channel activity and high concentrations of ADP (>300 μM) limited channel opening while GDP activated the channel at all concentrations tested. In the absence of internal Mg2+, ADP decreased channel activity at all concentrations tested while GDP had no noticeable effect at submillimolar concentrations and inhibited channel activity at millimolar concentrations. GDP [βS] (100 μM), which behaved as a weak GDP agonist in the presence of Mg2+, stimulated ADP-evoked activation whereas it inhibited GDP-evoked activation. The K+ channel opener pinacidil was found to activate the KATP channel but only in the presence of internal GDP, ADP and GDP [βS]. The results are discussed in terms of the existence of multiple nucleotide binding sites, in charge of the regulation of the KATP channel.

Control of intracellular calcium in the presence of nitric oxide donors in isolated skeletal muscle fibres from mouse

In skeletal muscle, nitric oxide (NO) is commonly referred to as a modulator of the activity of the ryanodine receptor (RyR) calcium release channel. However the reported effects of NO on isolated sarcoplasmic reticulum (SR) preparations and single ryanodine receptor (RyR) activity are diverse, and how NO affects SR calcium release and intracellular calcium homeostasis under physiological conditions remains poorly documented and hardly predictable. Here, we studied the effects of NO donors on membrane current and intracellular [Ca2+] in single skeletal muscle fibres from mouse, under voltage‐clamp conditions. When fibres were chronically exposed to millimolar levels of sodium nitroprusside (SNP) and challenged by short membrane depolarizations, there was a progressive increase in the resting [Ca2+] level. This effect was use‐dependent with the slope of rise in resting [Ca2+] being increased two‐fold when the depolarizing pulse level was raised from −20 to +10 mV. Analysis of the decay of the [Ca2+] transients suggested that cytoplasmic Ca2+ removal processes were largely unaffected by the presence of SNP. Also the functional properties of the dihydropyridine receptor were very similar under control conditions and in the presence of SNP. The resting [Ca2+] elevation due to SNP was accompanied by a depression of the peak calcium release elicited by pulses to +10 mV. The effects of SNP could be reproduced by the chemically distinct NO donor NOC‐12. They could be reversed upon exposure of the fibres to the thiol reducing agent dithiothreitol. Results suggest that large levels of NO produce a redox‐sensitive continuous leak of Ca2+ from the SR, through a limited number of release channels that do not close once they are activated by membrane depolarization. This SR Ca2+ leak and the resulting increase in resting [Ca2+] may be important in mediating the effects of excess NO on voltage‐activated calcium release.

Effects of extracellular ATP on freshly isolated mouse skeletal muscle cells during pre-natal and post-natal development.

Abstract. Extracellular adenosine 5′-triphosphate (ATP) has profound effects on membrane conductance and on the intracellular free [Ca2+] ([Ca2+]i) in cultured skeletal muscle cells. The aim of the present study was to examine the occurrence and to characterize the properties of such responses during mammalian muscle development in vivo. The effect of ATP (0.2xa0mM) was tested on membrane current and [Ca2+]i in freshly isolated pre- and post-natal mouse skeletal muscle cells. Pre-natal cells were from 14- to 19-day-old fetuses. In pre- and early post-natal cells, very small elevations of [Ca2+]i (<50xa0nM) following ATP application could be detected with the fluorescent indicator fura-2. A clear subsarcolemmal rise in [Ca2+] was however associated to the presence of ATP, as demonstrated by increased activity of plasma membrane Ca2+-activated K+ channels in cells bathed in a depolarizing, high-calcium-containing solution. In cells voltage-clamped at –80xa0mV in external Tyrode, ATP induced an inward current associated with an increased membrane conductance. The mean maximal amplitude of the ATP-induced current was –0.84±0.07xa0A/F (n=39). The response to ATP was still present after birth, although its amplitude tended to decrease with post-natal development and was completely absent in muscle cells from 3- to 6-month-old mice. The ATP-induced current could be abolished reversibly by suramin. Our results suggest that, over the range of developmental stages examined, skeletal muscle cells display an ionotropic purinergic signalling pathway with functional properties qualitatively consistent with what is observed in cultured myotubes.

Activation of Ca2+-activated K+ channels by an increase in intracellular Ca2+ induced by depolarization of mouse skeletal muscle fibres.

1 Ionic currents were simultaneously recorded at macroscopic and unitary level using the whole‐cell and cell‐attached patch‐clamp procedures together on the same portion of isolated mouse skeletal muscle fibres. 2 In the presence of Tyrode solution in the patch pipette and Tyrode‐TTX solution in the bath, macroscopic and unitary currents through delayed rectifier K+ channels were simultaneously recorded in response to depolarizing pulses of 1 s duration. 3 In five fibres, successive long‐lasting incremental depolarizing levels induced, at ‐40 mV or ‐30 mV, the opening of a high conductance channel carrying an outward current superimposed on delayed rectifier K+ channel activity. Opening of this high conductance channel was not observed when the depolarization steps were applied in the patch pipette. 4 Using the same depolarizing protocol, activation of a high conductance channel was also observed in two fibres in the presence of a K+‐rich solution in the pipette (145 mm K+). 5 With either Tyrode or K+‐rich solution in the pipette, unitary current amplitudes of the high conductance channel matched well with the values obtained for Ca2+‐activated K+ (KCa) channels in inside‐out patches under similar ionic conditions. 6 Indo‐1 fluorescence measurements showed that the stimulation protocol that led to KCa channel opening induced stepwise increases in intracellular [Ca2+] in the submicromolar range. 7 Our results provide evidence that activation of sarcolemmal KCa channels can be induced by a rise in intracellular [Ca2+] following voltage‐activated sarcoplasmic reticulum Ca2+ release.

Calcium signaling in isolated skeletal muscle fibers investigated under silicone voltage-clamp conditions

In skeletal muscle, release of calcium from the sarcoplasmic reticulum (SR) represents the major source of cytoplasmic Ca2+ elevation. SR calcium release is under the strict command of the membrane potential, which drives the interaction between the voltage sensors in the t-tubule membrane and the calcium-release channels. Either detection or control of the membrane voltage is thus essential when studying intracellular calcium signaling in an intact muscle fiber preparation. The silicone-clamp technique used in combination with intracellular calcium measurements represents an efficient tool for such studies. This article reviews some properties of the plasma membrane and intracellular signals measured with this methodology in mouse skeletal muscle fibers. Focus is given to the potency of this approach to investigate both fundamental aspects of excitation-contraction coupling and potential alterations of intracellular calcium handling in some muscle diseases.

Overexpression of transient receptor potential canonical type 1 (TRPC1) alters both store operated calcium entry and depolarization-evoked calcium signals in C2C12 cells

When the intracellular calcium stores are depleted, a Ca(2+) influx is activated to refill these stores. This store-operated Ca(2+) entry (SOCE) depends on the cooperation of several proteins as STIM1, Orai1, and, possibly, TRPC1. To elucidate this role of TRPC1 in skeletal muscle, TRPC1 was overexpressed in C2C12 cells and SOCE was studied by measuring the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)). TRPC1 overexpression significantly increased both the amplitude and the maximal rate-of-rise of SOCE. When YM-58483, an inhibitor of TRPC1 was used, these differences were eliminated, moreover, SOCE was slightly suppressed. A decrease in the expression of STIM1 together with the downregulation of SERCA was confirmed by Western-blot. As a consequence, a reduction in maximal Ca(2+) uptake rate and a higher resting [Ca(2+)](i) following the Ca(2+) transients evoked by 120mM KCl were detected. Morphological changes also accompanied the overexpression of TRPC1. Differentiation of the myoblasts started later, and the myotubes were thinner in TRPC1-overexpressing cultures. For these changes the observed decrease in the nuclear expression of NFAT1 could be responsible. Our results suggest that enhanced expression of TRPC1 increases SOCE and has a negative effect on the STIM1-Orai1 system, indicating an interaction between these proteins.

Expression of the muscular dystrophy-associated caveolin-3 P104L mutant in adult mouse skeletal muscle specifically alters the Ca 2+ channel function of the dihydropyridine receptor

Caveolins are plasma-membrane-associated proteins potentially involved in a variety of signalling pathways. Different mutations in CAV3, the gene encoding for the muscle-specific isoform caveolin-3 (Cav-3), lead to muscle diseases, but the underlying molecular mechanisms remain largely unknown. Here, we explored the functional consequences of a Cav-3 mutation (P104L) inducing the 1C type limb-girdle muscular dystrophy (LGMD 1C) in human on intracellular Ca2+ regulation of adult skeletal muscle fibres. A YFP-tagged human Cav-3P104L mutant was expressed in vivo in muscle fibres from mouse. Western blot analysis revealed that expression of this mutant led to an ∼80% drop of the level of endogenous Cav-3. The L-type Ca2+ current density was found largely reduced in fibres expressing the Cav-3P104L mutant, with no change in the voltage dependence of activation and inactivation. Interestingly, the maximal density of intramembrane charge movement was unaltered in the Cav-3P104L-expressing fibres, suggesting no change in the total amount of functional voltage-sensing dihydropyridine receptors (DHPRs). Also, there was no obvious alteration in the properties of voltage-activated Ca2+ transients in the Cav-3P104L-expressing fibres. Although the actual role of the Ca2+ channel function of the DHPR is not clearly established in adult skeletal muscle, its specific alteration by the Cav-3P104L mutant suggests that it may be involved in the physiopathology of LGMD 1C.