Acute exposure to extracellular BTP2 does not inhibit Ca²⁺ release during EC coupling in intact skeletal muscle fibers

N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2 or YM58483) is a Ca²⁺ release–activated Ca²⁺ (CRAC) channel inhibitor (Zitt et al., 2004) commonly used to assess the role of store-operated Ca²⁺ entry (SOCE). BTP2 inhibits SOCE activity through effects on both Orai1 and canonical transient receptor potential 3 (TRPC3) channels (He et al., 2005). Previous studies of SOCE in skeletal muscle cells have used 5–10 µM extracellular BTP2 to inhibit SOCE in intact myotubes (Eltit et al., 2013; Eltit et al., 2011; Li et al., 2010; Yarotskyy and Dirksen, 2012), acutely dissociated adult muscle fibers (Michelucci et al., 2019; Michelucci et al., 2020; Wei-LaPierre et al., 2013), and whole muscles (Boncompagni et al., 2017; Boncompagni et al., 2021). In the first issue of the Journal of General Physiology in 2021, Meizoso-Huesca and Launikonis (2021) published an intriguing study that reexamines the effect of BTP2 on Ca²⁺ handling in mechanically skinned rat extensor digitorum longus (EDL) muscle fibers. This study found that apart from its known ability to inhibit SOCE, BTP2 also inhibited both basal ryanodine receptor Ca²⁺ leak and evoked Ca²⁺ release (caused by either exposure to low intracellular Mg²⁺ or electrical stimulation). Specifically, the authors reported that both type 1 ryanodine receptor (RYR1) Ca²⁺ leak and Ca²⁺ release induced by exposure to low intracellular Mg²⁺, presumably due to activation of RYR1 in the SR, were reduced when 10 µM BTP2 was applied either intracellularly or in the extracellular solution (trapped within sealed transverse tubules [T-tubules] of the skinned fiber). While potential irreversible effects of intracellular BTP2 contamination that occur during mechanical skinning in the presence of BTP2 were not addressed, the authors concluded that BTP2 inhibits RYR1 channel activity regardless of the route of application. Importantly, Meizoso-Huesca and Launikonis (2021) also found that Ca²⁺ transients in skinned fibers elicited during repetitive (0.5 Hz) electrical twitch stimulation were essentially abolished following intracellular application of either 10 µM BTP2 (within ∼60 s) and 20 µM BTP2 (<10 s). The authors also provided evidence suggesting that this inhibition was unlikely the result of direct inhibition of RYR1 channels, but was dependent on the integrity of T-tubule and/or critical protein–protein interactions within the triad junction.

The results of Meizoso-Huesca and Launikonis (2021) have potentially important implications for interpreting results from prior studies that have used BTP2 as one means of assessing the relative role of SOCE in sustained Ca²⁺ transient amplitude (Michelucci et al., 2019; Wei-LaPierre et al., 2013) and contractile force production (Boncompagni et al., 2017; Boncompagni et al., 2021) during repetitive electrical stimulation. However, a significant limitation of this study is that the authors only tested the effect of intracellular applied BTP2 on electrically evoked Ca²⁺ release in mechanically skinned muscle fibers. Parallel studies on the effect of extracellular BTP2 on electrically evoked Ca²⁺ release in intact (or skinned) fibers were not completed. Such studies are critical since extracellular BTP2 is typically used to assess the relative role of SOCE on muscle physiology in intact myotubes, single fibers, and whole muscles, or even following systemic treatment of live animals (Zhao et al., 2012). Therefore, we determined the effect of extracellular BTP2 on electrically evoked RYR1 Ca²⁺ release during excitation–contraction (EC) coupling in single intact mouse muscle fibers following short-term exposure (2 min) using the same BTP2 concentrations and stimulation protocol employed by Meizoso-Huesca and Launikonis (2021). In addition, since BTP2 is highly lipophilic, the drug may cross the plasma membrane slowly over time to reach intracellular compartments when applied extracellularly. Therefore, we also examined the effect of prolonged exposure of extracellular BTP2 (30 min) in intact muscle fibers. The results provide important concentration and exposure guidance with regard to the proper use of BTP2 to inhibit preferentially SOCE in intact skeletal muscle fibers.

All experiments used 4-mo-old male and female C57BL6/J wild-type mice. Mice were group-housed in sterile ventilated microisolator cages on corncob bedding in the University of Rochester Medical Center vivarium, an Association for Assessment and Accreditation of Laboratory Animal Care International–accredited facility. All animal procedures were approved by the University Committee on Animal Resources at the University of Rochester.

All chemicals and dyes were purchased from Sigma-Aldrich or Invitrogen. Stock concentrations of mag-fluo-4-AM (1 mM) and BTP2 (10 mM) were made up in DMSO and then diluted to their respective final concentrations in Ringer’s solution consisting of (in mM) 146 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, and 10 HEPES, pH 7.4). The efficiency of BTP2 to block SOCE at the concentrations used in this study was validated in Mn²⁺ quench studies as described previously (Michelucci et al., 2019; Wei-LaPierre et al., 2013).

Acutely dissociated FDB fibers were isolated from mouse footpads before experiments using enzymatic digestion as previously described (Lueck et al., 2007). Single intact FDB fibers were plated on glass-bottom dishes and loaded with low-affinity Ca²⁺ dye, mag-fluo-4 (4 µM), in Ringer’s solution for 20 min at room temperature. Following 20 min incubation in dye-free Ringer’s supplemented with 25 µM N-benzyl-p-toluene sulfonamide, a skeletal muscle myosin inhibitor to reduce movement artifacts during stimulation, fibers were mounted on the stage of a Nikon Eclipse 2000E inverted microscope. Twitch electrical stimulation was delivered to individual FDB fibers using a NaCl-filled glass electrode placed adjacent to the cell of interest. For short-term BTP2 exposure, FDB fibers were stimulated at a frequency of 0.5 Hz for a total of 3 min, where control Ringer’s was perfused during the initial 60 s followed by 2 min in Ringer’s solution supplemented with DMSO vehicle, 10 µM BTP2, or 20 µM BTP2. For BTP2 application, a local perfusion system that enabled fast and complete solution exchange within 5 s was employed with a perfusion barrel placed adjacent to the fiber of interest. Throughout the stimulation period, mag-fluo-4 fluorescence within a selected portion of the fiber interior was excited at 480 ± 15 nm using an Excite epifluorescence illumination system (Nikon Instruments). Fluorescence emission at 535 ± 30 nm was monitored using a 40× oil objective and photomultiplier detection system (Photon Technologies Inc.). For prolonged BTP2 exposure, FDB fibers were continuously stimulated at a frequency of 0.1 Hz for 30 min in the presence of DMSO vehicle, 10 µM BTP2, or 20 µM BTP2. During the stimulation period, mag-fluo-4 fluorescence was acquired for four successive transients every 5 min, while keeping the computer-controlled shutter closed in between data collection in order to minimize fiber phototoxicity and photobleaching of the dye. Relative changes in mag-fluo-4 fluorescence from baseline (ΔF/F₀) were digitized at 10 kHz and analyzed using Clampfit 10.0 (Molecular Devices) and Igor (Wave Metrics) software.

The peak change in ΔF/F₀ during electrical stimulation was determined for each twitch Ca²⁺ transient throughout the 3-min period of short-term BTP2 exposure and averaged across the four successive twitch Ca²⁺ transients acquired at 5-min intervals during long-term BTP exposure. Kinetic analyses of the rise and decay phases of the transients elicited during short-term exposure were assessed from the average of the last five transients in control Ringer’s solution (before addition of DMSO or BTP2) and the final five transients after 2 min exposure to either DMSO or BTP2.

All data groups were verified to follow a normal distribution before conducting parametric statistical analyses using the Prism (GraphPad) software. One-way ANOVA with post hoc Tukey test was used to statistically compare peak amplitude data. Two-way ANOVA with post hoc Tukey test was used for statistical analysis of the kinetic components of Ca²⁺ transient. All data were expressed as mean ± SEM with P < 0.05 considered statistically significant.

Prior studies used 10 µM BTP2 to inhibit SOCE in intact fibers (Michelucci et al., 2019; Michelucci et al., 2020; Wei-LaPierre et al., 2013) and whole muscles (Boncompagni et al., 2017; Boncompagni et al., 2021). Using the stimulation paradigm of Meizoso-Huesca and Launikonis (2021), extracellular application of 10 µM BTP2 did not significantly alter the magnitude of electrically evoked Ca²⁺ release compared with control even after 2 min (peak ΔF/F₀: 1.02 ± 0.04 for DMSO and 1.02 ± 0.04 for 10 µM BTP2; Fig. 1, A, B, and D). In fact, electrically evoked Ca²⁺ transient amplitude was also not significantly altered 2 min after exposure to even 20 µM BTP2 (peak ΔF/F₀: 0.93 ± 0.06 for 20 µM BTP2; Fig. 1, C and D). As a result, peak Ca²⁺ transient amplitudes were not significantly different for any time point across all groups throughout the entire 3-min stimulation period (Fig. 1 D).

Superimposed traces from FDB fibers before (Fig. 2 A) and 2 min after (Fig. 2 B) treatment with vehicle (DMSO), 10 µM, and 20 µM BTP2 exhibited a similar rate of rise. Similarly, the rate of rise of the transient was also comparable in FDB fibers before and after exposure for 2 min to DMSO (Fig. 2 C), 10 µM (Fig. 2 D), or 20 µM BTP2 (Fig. 2 E). To better quantify this, the maximum rate of electrically evoked Ca²⁺ release was approximated from the peak dF/dt of the mag-fluo-4 fluorescence. This analysis revealed that peak dF/dt was not significantly different between FDB fibers 2 min after exposure to vehicle control (DMSO), 10 µM, or 20 µM BTP2 (Fig. 3 D).

In addition, the transient decay kinetics were also similar for all conditions in control (Fig. 2 F) and 2 min after exposure (Fig. 2 G). The decay phase of the electrically evoked mag-fluo-4 transients is well-described by a double exponential fit where the fast component of decay primarily reflects Ca²⁺ binding to fast myoplasmic Ca²⁺ buffers and the slow component of decay is dominated by SERCA pump-mediated reuptake (Baylor and Hollingworth 2003; Capote et al., 2005; Carroll et al., 1999). Thus, we fit the decay phase of the transients with a double exponential equation to quantify the fast (A_fast and τ_fast) and slow (A_slow and τ_slow) components before and after treatment with vehicle, 10 µM, and 20 µM BTP2 (representative traces and corresponding double exponential fits shown in Fig. 3, A–C). No statistically significant differences in the fast or slow amplitudes of decay (Fig. 3 E), the fast time constant of decay (Fig. 3 F), or the slow time constant of decay (Fig. 3 G) were observed between fibers exposed to DMSO (vehicle), 10 µM BTP2, or 20 µM BTP2. While some time-dependent differences (e.g., between 0 and 2 min) were observed in the fast amplitude of decay, these were not pursued further as the changes were similar across all treatment groups. The above results indicate that the magnitude and kinetics of electrically evoked Ca²⁺ release during EC coupling in intact FDB fibers is not significantly altered following short-term (2 min) exposure to either 10 µM BTP2 or 20 µM BTP2.

Given the highly lipophilic properties of BTP2, prolonged extracellular exposure would be expected to result in an initial partition into surface and T-tubule membranes followed by slower diffusion across the sarcolemma to nearby intracellular compartments. To investigate this possibility, we examined the effect of prolonged exposure to DMSO, 10 µM BTP2, or 20 µM BTP2 (up to 30 min) on electrically evoked Ca²⁺ transients in intact FDB fibers. For these studies, FDB fibers were stimulated at a lower frequency (0.1 Hz), and mag-fluo-4 fluorescence was monitored for several successive transients every 5 min with the shutter closed in between recording intervals (to minimize phototoxicity/bleaching). Extracellular application of 10 µM BTP2 did not significantly alter peak Ca²⁺ transient amplitude from that of vehicle control (DMSO) until after 25 min of exposure (Fig. 4, blue traces and blue bars). In contrast, peak Ca²⁺ transient amplitude was significantly reduced as early as 5 min after exposure to 20 µM BTP2, which then continued to decline in a time-dependent manner, reaching ∼20% of DMSO control after 30 min (Fig. 4, red traces and red bars).

The primary finding of this communication is that electrically evoked Ca²⁺ release in intact mouse FDB fibers is unaltered by extracellular application of 10 µM BTP2 for up to 25 min (Figs. 1, 2, and 3). These findings indicate that EC coupling is unaltered by BTP2 exposure conditions used previously to inhibit SOCE in intact muscle preparations (Boncompagni et al., 2017; Boncompagni et al., 2021; Eltit et al., 2013; Li et al., 2010; Yarotskyy and Dirksen, 2012; Zhao et al., 2012; Eltit et al., 2011; Wei-LaPierre et al., 2013; Michelucci et al., 2019; Michelucci et al., 2020). Moreover, these results are also consistent with prior findings that SOCE does not significantly contribute to Ca²⁺ transients during EC coupling (Koenig et al., 2018; Meizoso-Huesca and Launikonis, 2021).

Consistent with these results, we previously reported that inhibition of SOCE by exposure to 10 µM BTP2 for 2.5–10 min does not alter peak electrically evoked Ca²⁺ release in intact FDB fibers (Wei-LaPierre et al., 2013) or peak specific force production in intact muscle (Michelucci et al., 2019) during a single high-frequency stimulation train. Similar results were observed following inducible, muscle-specific Orai1 knockout (Carrell et al., 2016; Michelucci et al., 2019). However, inhibition of SOCE increases fatigability during repetitive high-frequency stimulation, which is reflected in both a reduction in Ca²⁺ transient amplitude following short-term exposure (<10 min) to 10 µM BTP2 (Wei-LaPierre et al., 2013) and muscle-specific force production following inducible ablation of Orai1 (Michelucci et al., 2019). These studies used the same stimulation paradigm (500-ms stimulations delivered at 50 Hz, repeated every 2.5 s) and verified that inhibition of SOCE (both pharmacologically and genetically) results in a comparable increase in fatigue during repetitive high-frequency stimulation. The finding in this communication supports these previous studies by demonstrating that short-term exposure (<30 min) of intact FDB fibers to 10 µM BTP2 does not significantly affect Ca²⁺ release during EC coupling. Thus, effects of acute exposure in intact fibers/muscle up to 10 µM BTP2 reported previously are not due to effects of the drug on RYR1-mediated Ca²⁺ release during EC coupling.

While Ca²⁺ release during EC coupling is not reduced following short-term exposure to 10 µM BTP2, it is reduced for longer exposures to 10 µM BTP2 (>30 min) and for shorter exposures to 20 µM BTP2 (>5 min). While peak Ca²⁺ transient amplitude is reduced in the presence of 20 µM BTP2 after ∼30 action potentials at 0.1 Hz (5 min, Fig. 4), it is unaltered at this concentration after 60 action potentials applied at a higher frequency over a shorter period of time (0.5 Hz in 2 min, Fig. 1). These results indicate that the inhibitory effect of BTP2 on Ca²⁺ release during EC coupling is primarily concentration- and time-dependent rather than use-dependent, consistent with delayed access to intracellular compartments when applied to intact fibers. Together, our studies provide critical evidence to support the use of 10 µM BTP2 for up to 25 min for physiological studies of SOCE activity in intact muscle fibers.

Our findings of the absence of any effect of 2 min exposure to 10–20 µM on EC coupling in intact muscle fibers (Fig. 1) are in marked contrast to those reported by Meizoso-Huesca and Launikonis (2021) in mechanically skinned rat EDL muscle fibers. In skinned fibers, intracellular application of 10 µM BTP2 reduced electrically evoked Ca²⁺ release (0.5 Hz stimulation) ∼50% after 20–25 s, and 20 µM BTP2 essentially abolished Ca²⁺ release in <10 s. The results of our prolonged BTP2 exposure experiments in intact fibers (Fig. 4) suggest that the discrepancy between our results and those of Meizoso-Huesca and Launikonis (2021) is most likely due to the method of BTP2 application (extracellular versus intracellular). However, potential differences in species (mouse versus rat), muscle (FDB versus EDL), and experimental paradigms (intact versus skinned fibers) used cannot be excluded. Whatever the reason, our results demonstrate that caution needs to be taken when extrapolating effects of agents applied to the intracellular solution in skinned fiber preparations compared with extracellular application of the same agent in intact fibers.

Meizoso-Huesca and Launikonis (2021) proposed that BTP2 inhibition of RYR1 was independent of whether the compound was applied intracellularly or extracellularly as Ca²⁺ release induced during exposure to low intracellular Mg²⁺ was similarly reduced regardless of the orientation of BTP2 application. However, the inhibition of electrically evoked Ca²⁺ release by intracellular application of BTP2 reported by Meizoso-Huesca and Launikonis (2021) was not repeated under conditions when BTP2 application was restricted to the extracellular space. As BTP2 is highly lipophilic, the compound preferentially partitions into membranes. When applied extracellularly, the drug will primarily partition into sarcolemmal and T-tubule membranes. Our results observed following prolonged exposure of intact fibers to BTP2 support the possibility that the compound may only slowly cross sarcolemmal and T-tubule membranes and diffuse into intracellular membranes during prolonged exposure. It is unclear in the study by Meizoso-Huesca and Launikonis (2021) precisely how long BTP2 was trapped in the T-tubule before the Ca²⁺ measurements were made. In addition, potential local intracellular accumulation of BTP2 cannot be excluded in these studies since the previously applied drug was present during mechanical skinning.

In summary, our results validate the use of 10 µM BTP2 for up to 25 min as a means of preferentially inhibiting SOCE in intact mouse FDB fibers in the absence of a significant impact on Ca²⁺ release during EC coupling.

Eduardo Ríos served as editor.

This work was supported by a grant from the National Institutes of Health (AR059646 to R.T. Dirksen).

The authors declare no competing financial interests.

Author contributions: L. Wei-LaPierre, conceptualization, data curation, formal analysis, visualization, and writing–original draft, review, and editing; L. Groom, data curation, formal analysis, investigation, project administration, visualization, and writing–review and editing; and R.T. Dirksen, conceptualization, funding acquisition, supervision, visualization, and writing–original draft, review, editing.