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Tight control of skeletal muscle contractile activation is secured by the excitation–contraction (EC) coupling protein complex, a molecular machinery allowing the plasma membrane voltage to control the activity of the ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. This machinery has been shown to be intimately linked to the plasma membrane protein pannexin-1 (Panx1). We investigated whether the prescription drug probenecid, a widely used Panx1 blocker, affects Ca2+ signaling, EC coupling, and muscle force. The effect of probenecid was tested on membrane current, resting Ca2+, and SR Ca2+ release in isolated mouse muscle fibers, using a combination of whole-cell voltage-clamp and Ca2+ imaging, and on electrically triggered contraction of isolated muscles. Probenecid (1 mM) induces SR Ca2+ leak at rest and reduces peak voltage-activated SR Ca2+ release and contractile force by 40%. Carbenoxolone, another Panx1 blocker, also reduces Ca2+ release, but neither a Panx1 channel inhibitory peptide nor a purinergic antagonist affected Ca2+ release, suggesting that probenecid and carbenoxolone do not act through inhibition of Panx1-mediated ATP release and consequently altered purinergic signaling. Probenecid may act by altering Panx1 interaction with the EC coupling machinery, yet the implication of another molecular target cannot be excluded. Since probenecid has been used both in the clinic and as a masking agent for doping in sports, these results should encourage evaluation of possible effects on muscle function in treated individuals. In addition, they also raise the question of whether probenecid-induced altered Ca2+ homeostasis may be shared by other tissues.

International audience; The zebrafish has emerged as a very relevant animal model for probing the pathophysiology of human skeletal muscle disorders. This vertebrate animal model displays a startle response characterized by high-frequency swimming activity powered by contraction of fast skeletal muscle fibers excited at extremely high frequencies, critical for escaping predators and capturing prey. Such intense muscle performance requires extremely fast properties of the contractile machinery but also of excitation–contraction coupling, the process by which an action potential spreading along the sarcolemma induces a change in configuration of the dihydropyridine receptors, resulting in intramembrane charge movements, which in turn triggers the release of Ca2+ from the sarcoplasmic reticulum. However, thus far, the fastest Ca2+ transients evoked by vertebrate muscle fibers has been described in muscles used to produce sounds, such as those in the toadfish swim bladder, but not in muscles used for locomotion. By performing intracellular Ca2+ measurements under voltage control in isolated fast skeletal muscle fibers from adult zebrafish and mouse, we demonstrate that fish fast muscle fibers display superfast kinetics of action potentials, intramembrane charge movements, and action potential–evoked Ca2+ transient, allowing fusion and fused sustained Ca2+ transients at frequencies of excitation much higher than in mouse fast skeletal muscle fibers and comparable to those recorded in muscles producing sounds. The present study is the first demonstration of superfast kinetics of excitation–contraction coupling in skeletal muscle allowing superfast locomotor behaviors in a vertebrate.

This article introduces a control method for switched-capacitor (SC) converters used directly or indirectly in emerging point-of-load (PoL) applications. Compared to conventional methods of controlling SC converters relying on the simple transformer model, the introduced method takes into account system dynamics and improves transient response by effectively reducing the order of the system. This is achieved by controlling the flying capacitor voltage, eliminating its dynamics. The improvement in transient response results in lower output voltage deviation than conventional methods, allowing a reduction in the size of the output capacitor. In addition, this improvement in transient response allows for the elimination of low drop out regulators (LDOs) in some cascaded PoL systems. It has been shown that the inner loop controlling the flying capacitor can be implemented explicitly controlling the top and bottom bounds of the flying capacitor voltage. The advantages of the control method have been demonstrated with mixed-signal experimental results for a 1-W cascaded 4:1 SC converter and a 200-mW 2:1 SC converter and comparative simulations. Advantages compared with output voltage observation only have been verified, varying from a 50% reduction in output voltage deviation in the worst case to no voltage deviation in the best case.

Pannexins are plasma membrane heptameric channels mediating ATP release from the cytosol to the extracellular space. Skeletal muscle activity is associated with Pannexin 1 (Panx1) channels activation, ATP release out to the extracellular space and subsequent activation of purinergic signaling pathways. In agreement, recent evidence has shown molecular and functional interactions between Panx1 and the excitation–contraction (EC) coupling machinery of skeletal muscle. In this framework, we tested whether pharmacological effectors of Panx1 affect EC coupling in differentiated muscle fibers. Using confocal detection of cytosolic Ca2+ in voltage-clamped mouse muscle fibers, we found that the Panx1 blocker probenecid (1 mM) affects intracellular Ca2+ handling and EC coupling: acute application of probenecid generates a rise in resting Ca2+ that also occurs in nominally Ca2+-free extracellular medium. This effect is associated with a reduction of Ca2+ release through the sarcoplasmic reticulum (SR) Ca2+ channel RYR1. The effect of probenecid persists with time, with muscle fibers incubated for 30 min in the presence of the drug exhibiting a 40% reduction in peak SR Ca2+ release. Under the same conditions, the other Panx1 blocker carbenoxolone (50 µM) produced a 70% reduction in peak SR Ca2+ release. Application of probenecid on electrically stimulated whole mouse muscle induced a slight rise in resting tension and a >50% reduction of tetanic force after 30 min of incubation. Our results provide further support for the strong links between Panx1 function and EC coupling. Because probenecid is used both in the clinic for several types of therapeutic benefits and as a hiding agent for doping in sport, our results question whether potential adverse muscular effects may have, so far, been overlooked.

The zebrafish has emerged as a very relevant animal model to decipher the pathophysiology of human muscle disorders. However, the vast majority of studies on zebrafish skeletal muscle have investigated genetic, histological, and molecular aspects, but functional approaches at the cellular level, especially in the field of excitation–contraction (EC) coupling, are scarcer and generally limited to cultured myotubes or fibers from embryonic zebrafish. Considering that zebrafish undergoes profound metamorphosis during transition from larval to adult stage and that number of muscle pathologies come up at ages far beyond embryonic stages, there is an actual need to investigate EC coupling in fully differentiated zebrafish skeletal muscle. In the present study, we were able to implement current and voltage clamp combined with intracellular Ca2+ measurements using the intracellularly loaded Ca2+ dye indo-1 in enzymatically isolated fast skeletal muscle fibers from 1-yr old zebrafish. Recording of action potentials (AP) in current-clamp conditions revealed very fast kinetics of the repolarization phase of AP. Measurements of intramembrane charge movements in voltage-clamp conditions showed that charge movement density was half that measured in mammalian fibers, but they displayed much faster kinetics. Ca2+ transients elicited by depolarization displayed a voltage-dependent phase of activation and voltage- and time-dependent phase of inactivation. Recording of Ca2+ signals elicited by trains of AP at different rates in current-clamp conditions indicated that Ca2+ signals fused at very high stimulation frequencies with no sign of Ca2+ signal decay for the entire 0.5 s duration of the stimulation, giving evidence that fibers were still able to generate AP and the sarcoplasmic reticulum to release Ca2+ with stimulation rates as high as 200 Hz. These data indicate that adult zebrafish fast skeletal muscle fibers exhibit strikingly fast kinetics of EC coupling from AP firing to charge movements and sarcoplasmic reticulum Ca2+ release.