BTP2 negatively regulates Orai1 and ryanodine receptor function in skeletal muscle

Background. BTP2 is known to block Orai1, the Ca2+ channel of store-operated Ca2+ entry (SOCE) but no detailed analysis has been undertaken in skeletal muscle, where the drug has been used extensively to study SOCE. Methodology. We trapped a Ca2+ sensitive dye in the tubular (t-) system of mechanically skinned fibres from rat to define the effect of BTP2 on SOCE in skeletal muscle fibres and used a cytoplasmic rhod-2 to track Ca2+ release in the presence of BTP2. Results. In addition to blocking Orai1-dependent SOCE, we found a BTP2-dependent inhibition of a resting Ca2+ conductance, likely to be through the Orai1 channel. Intriguingly, increasing concentrations of BTP2 displayed a hormetic effect on resting [Ca2+] in the t-system ([Ca2+]t−sys), shifting from inducing an accumulation of Ca2+ in the t-system presumably due to Orai1 channels blocking, to reducing the resting [Ca2+]t−sys. In absence of functional ryanodine receptors (RyRs), this biphasic effect was not observed, suggesting that above the hormetic zone, BTP2 impairs RyR function. Additionally, we found that BTP2 impairs the cytoplasmic Ca2+ transients during repetitive excitation-contraction coupling (EC coupling) cycles, independently of extracellular Ca2+ entry impairment. We determined that the release of Ca2+ through the RyR was inhibited by BTP2, strongly suggesting that the RyR was the point of inhibition during the cycles of EC coupling. Finally, we found that BTP2 inhibition of RyR-mediated Ca2+ release was independent of extracellular or intracellular application of the agent, indicating that BTP2 can impair RyR function in intact muscle. Conclusion. Our results show that both Ca2+ channels, the Orai1 and RyR, are negatively regulated by BTP2, shedding new light on previous work that applied BTP2 to block SOCE in muscle.

BTP2 negatively regulates Orai1 and ryanodine receptor function in skeletal muscle

Background. BTP2 is known to block Orai1, the Ca 2+ channel of store-operated Ca 2+ entry (SOCE) but no detailed analysis has been undertaken in skeletal muscle, where the drug has been used extensively to study SOCE. Methodology. We trapped a Ca 2+ sensitive dye in the tubular (t-) system of mechanically skinned fibres from rat to define the effect of BTP2 on SOCE in skeletal muscle fibres and used a cytoplasmic rhod-2 to track Ca 2+ release in the presence of BTP2. Results. In addition to blocking Orai1-dependent SOCE, we found a BTP2-dependent inhibition of Orai1 channel resting Ca 2+ conductance. Intriguingly, increasing concentrations of BTP2 displayed a hormetic effect on resting [Ca 2+ ] in the t-system ([Ca 2+ ] t-sys ), shifting from inducing an accumulation of Ca 2+ in the t-system presumably due to Orai1 channels blocking, to reducing the resting [Ca 2+ ] t-sys . This biphasic effect is not observed in presence of a ryanodine receptor (RyR) inhibitor, suggesting that above the hormetic zone, BTP2 impairs RyR function. Additionally, we found that BTP2 impairs the cytoplasmic Ca 2+ transients during repetitive excitation-contraction coupling (EC coupling) cycles independent of extracellular Ca 2+ entry. We determined that the release of Ca 2+ through the RyR was inhibited by BTP2, strongly suggesting that the RyR was the point of inhibition during the cycles of EC coupling. Conclusion. Our results show that both Ca 2+ channels, the Orai1 and RyR, are negatively regulated by BTP2, shedding new light on previous work that applied BTP2 to block SOCE in muscle.

Diaphragm tension reduced in dystrophic mice by an oxidant, hypochlorous acid

In dystrophin-deficient skeletal muscle cells, in which Ca2+ homeostasis is disrupted and reactive oxygen species production is increased, we hypothesized that hypochlorous acid (HOCl), a strong H2O2-related free radical, damages contractile proteins and the sarcoplasmic reticulum. The aim of the present study was to investigate the effects of exposure to oxidative stress, generated by applying HOCl (100 µmol/L and 1 mmol/L), on the contractile function and sarcoplasmic reticulum properties of dystrophic mice. Experiments were performed on diaphragm muscle, which is severely affected in the mdx mouse, and the results were compared with those obtained in healthy (non-dystrophic) mice. In Triton-skinned fibres from C57BL/10 and mdx mice, 1 mmol/L HOCl increased myofibrillar Ca2+ sensitivity, but decreased maximal Ca2+-activated tension. In the presence of HOCl, higher concentrations of MgATP were required to produce rigor tensions. The interaction between HOCl and the Ca2+ uptake mechanisms was demonstrated using saponin-skinned fibres and sarcoplasmic reticulum vesicles. The results showed that HOCl, at micromolar or millimolar concentrations, can modify sarcoplasmic reticulum Ca2+ uptake and that this effect was more pronounced in diaphragm muscle from mdx mice. We conclude that in dystrophic diaphragm skeletal muscle cells, HOCl activates a cellular pathway that leads to an increase in the intracellular concentration of Ca2+.

Adenosine reduces the reverse mode of the Na+/Ca2+ exchanger in ferret cardiac fibres

The aim of this study was to investigate the effects of adenosine on reverse mode Na+/Ca2+ exchange. In intact ferret cardiac trabeculae, Na+-free contractures were investigated after treating preparations with ryanodine, a sarcoplasmic reticulum Ca2+-channel inhibitor, and thapsigargin, a sarcoplasmic reticulum Ca2+-pump inhibitor added to suppress the sarcoplasmic reticulum function. The effects of adenosine (50–100 nmol/L), adenosine deaminase (ADA, 0.1–0.5 U/L), the A1 and A2A receptor agonists CCPA (3–100 nmol/L) and CGS 21680 (25–100 nmol/L), and the A1 and A2A receptor antagonists DPCPX (25 nmol/L) and ZM 241385 (25 nmol/L) were tested on Na+-free contractures. The application of adenosine (50–100 nmol/L) had no significant effect on the characteristics of the Na+-free contractures. However, the results show that treatment with ADA (0.3 U/L), adenosine (≥50 nmol/L) and CCPA, a specific A1 receptor agonist (3–100 nmol/L), all reduced the Na+-free contracture amplitude. In the presence of ADA, the effects of adenosine and CCPA were also reduced by a specific antagonist of A1 receptors (DPCPX, 25 nmol/L). Furthermore, adenosine, ADA, and CCPA did not affect the properties of the contractile apparatus in Triton-skinned fibres. It is therefore proposed that endogenous adenosine reduced the reverse mode of the Na+/Ca2+ exchanger by acting on A1 receptors present in the sarcolemmal membrane.

Determinants of surface membrane and transverse-tubular excitability in skeletal muscle: implications for high-intensity exercise

The fatigue of high-intensity exercise is now believed to reside primarily within the excitation–contraction coupling processes associated with the plasma membrane of skeletal muscle (sarcolemm) and calcium-mediated events leading to myofilament sliding. This paper summarizes recent developments and advances in the identification of factors that contribute to changes in sarcolemmal excitability of mammalian skeletal muscle as a consequence of high-intensity exercise. There is an increasing recognition of the probable role that is played by the transverse tubular system (T-system), a system that comprises c. 80% of the total sarcolemmal surface capable of ion exchange. Furthermore, the fluid within the T-system has limited access to interstitial fluid bathing myofibres; hence, T-system fluid is probably markedly different from interstitial fluid during high-intensity exercise. Mechanically skinned fibre preparation is providing many new insights into functions of the surface membrane and T-system in fatigue. A scenario is developed whereby accumulation of potassium within the T-system ([K+]o) contributes to reduced membrane excitability, as well as lowering of T-system sodium and chloride, concomitant with loss of intracellular potassium ([K+]i) and accumulation of intracellular sodium ([Na+]) and chloride ([Cl−]). Lowering the [Na+]o/[Na+]i ratio and raising myoplasmic [Na+]i have been shown to decrease membrane excitability and impair action potential propagation. Maintained high [Cl−]o may also have a protective effect in maintaining membrane excitability, and this effect appears to be very pronounced in the presence of raised [K+]o. In contrast to dogma associating high [H+] to fatigue, recent studies have also shown that induced acidosis that results in increased [H+]o and [H+]i restores force production in muscles and skinned fibres fatigued by intermittent tetanic stimulation. This effect may be due to a decrease in surface membrane Cl− permeability that serves to restore membrane excitability. During high-intensity exercise, simultaneous changes in trans-membrane ion concentrations and membrane ion conductances may serve to reduce impairment of membrane excitability that provides for a maintained, though reduced, contractile function.