Triclosan impairs excitation-contraction coupling and Ca2+ dynamics in striated muscle

Abstract

Triclosan (TCS), a high-production-volume chemical used as a bactericide in personal care products, is a priority pollutant of growing concern to human and environmental health. TCS is capable of altering the activity of type 1 ryanodine receptor (RyR1), but its potential to influence physiological excitation-contraction coupling (ECC) and muscle function has not been investigated. Here, we report that TCS impairs ECC of both cardiac and skeletal muscle in vitro and in vivo. TCS acutely depresses hemodynamics and grip strength in mice at doses ≥12.5 mg/kg i.p., and a concentration ≥0.52 μM in water compromises swimming performance in larval fathead minnow. In isolated ventricular cardiomyocytes, skeletal myotubes, and adult flexor digitorum brevis fibers TCS depresses electrically evoked ECC within ∼10-20 min. In myotubes, nanomolar to low micromolar TCS initially potentiates electrically evoked Ca(2+) transients followed by complete failure of ECC, independent of Ca(2+) store depletion or block of RyR1 channels. TCS also completely blocks excitation-coupled Ca(2+) entry. Voltage clamp experiments showed that TCS partially inhibits L-type Ca(2+) currents of cardiac and skeletal muscle, and [(3)H]PN200 binding to skeletal membranes is noncompetitively inhibited by TCS in the same concentration range that enhances [(3)H]ryanodine binding. TCS potently impairs orthograde and retrograde signaling between L-type Ca(2+) and RyR channels in skeletal muscle, and L-type Ca(2+) entry in cardiac muscle, revealing a mechanism by which TCS weakens cardiac and skeletal muscle contractility in a manner that may negatively impact muscle health, especially in susceptible populations.

Fig. 1.

TCS depresses hemodynamics and in vivo skeletal muscle function. (A) Mice (vehicle: n = 3; TCS: n = 4–5) were anesthetized with ketamine/xylazine and instrumented with recording PV catheter advanced into the left ventricle via the carotid artery. After dosing with TCS (20% DMSO vol/vol in 200 μL) intraperitoneally, both 12.5 and 25 mg/kg groups experienced significant reductions in cardiac output, ventricular filling, and left ventricular developed pressure. Error bars represent SEM. *P < 0.05; **P < 0.01, one-tailed paired t test. (B) Grip strength was assessed in mice (n = 7 per group) before and after sham, vehicle (30 μL DMSO), or 40 mg/kg TCS intraperitoneal injection. Postdose grip was significantly lower in the TCS group compared with both sham and vehicle. Gray bars represent mean and 95% CI. **P < 0.01, ANOVA. (C) Larval fathead minnow (n = 36 per concentration) were exposed up to 7 d to vehicle (0.01% MeOH), or TCS (0.035, 0.26, or 0.52 μM). Swimming behavior was assessed by nonprovoked (distance traveled, bar graph) and forced (lines crossed in 60 s, line graph) activity. A decreasing trend was seen for both swimming parameters, with significance found in the 0.52-μM group. Error bars represent SEM. **P < 0.01, Kruskal-Wallis.

Fig. 2.

TCS impairs cardiac ECC. (A) Isolated mouse cardiomyocytes (n = 5) were loaded with the fluorescent Ca²⁺ indicator Fluo-4. RyR2-mediated Ca²⁺ transients were evoked by field stimulation and measured by line-scan confocal imaging. Twenty minutes after applying 10 μM TCS, Ca²⁺ transient amplitudes were significantly reduced by 79.3 ± 5.2%. (B) Cardiomyocytes (n = 5) were patch-clamped in whole-cell configuration, and depolarized by voltage protocol. Exposure to 10 μM TCS for 20 min significantly reduced peak inward currents through Ca_v1.2 by 54.4 ± 9.6% and prolonged the inactivation time constant from 25.8 ± 2.0 ms to 45.2 ± 3.6 ms. All error bars represent SEM. **P < 0.01; ***P < 0.001, t test.

Fig. 3.

TCS impairs skeletal ECC. (A) Mouse skeletal myotubes were loaded with the Ca²⁺ indicator Fluo-4 and electrically stimulated. Compared with vehicle (0.1% DMSO; Top) perfusion, 0.5 μM TCS significantly potentiated Ca²⁺ transient amplitudes (Middle). A similar potentiation was initially seen with 1 μM TCS, but this invariably led to the diminution and complete abrogation of electrically evoked Ca²⁺ transients (Bottom). (B) The effect of TCS on ECC is highly dose-dependent, with 10 μM causing rapid ECC failure within 12.5 min. All TCS exposures significantly altered the Ca²⁺ transient amplitude responses to single electrical test pulses (P < 0.01). Error bars represent SEM. (C) Exposure to submicromolar TCS for 24 h was sufficient to alter transient amplitudes in myotubes, with 0.5 μM TCS producing significant reductions at 5–40 Hz stimulus frequencies. Error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001, two-way ANOVA.

Fig. 6.

TCS concomitantly affects both RyR and DHPR activity without depleting SR Ca²⁺ stores. (A) Mouse skeletal myotubes were loaded with the Ca²⁺ indicator Fluo-4 and electrically stimulated. Perfusion with 10 μM TCS resulted in a profound change in resting Ca²⁺, as well as a rapid diminution and abrogation of Ca²⁺ transients. Challenge with 20 mM caffeine (Caff) indicates SR Ca²⁺ stores were not depleted. (Inset) In separate experiments, after TCS caused failure of myotubes to respond to electrical stimuli, challenge with 60 mM KCl (K⁺) also failed to elicit ECC. (B) Dyspedic myotubes lacking RyR1 exhibited little change in cytosolic Ca²⁺ when exposed to TCS, implicating RyR1 as a molecular target of TCS. (C) Mouse skeletal muscle preparations were incubated with [³H]ryanodine or [³H]PN200 in the presence of TCS. TCS increased specific [³H]ryanodine binding with respect to control, whereas specific [³H]PN200 binding was inhibited noncompetitively across similar TCS concentrations.

Fig. 4.

TCS attenuates skeletal muscle L-type Ca²⁺ current. (A) Averaged recordings of L-type Ca²⁺ currents elicited by 2-s depolarizations from −50 mV to +30 mV are shown for untreated normal myotubes (black circle, n = 4) or normal myotubes exposed to 10 μM TCS for 15–20 min at ∼25 °C (gray circle, n = 9). (B) Summary of peak current densities (Left) and r₂ values (Right) for myotubes in the presence and absence of 10 μM TCS. r₂ is ratio of the current remaining at 2 s to the peak current. Error bars represent SEM. ***P < 0.001, t test.

Fig. 5.

TCS inhibits excitation-coupled Ca²⁺ entry. (A) The rate of ECCE was measured using Mn²⁺ quench of Fura-2 fluorescence at the isosbestic wavelength. Electrical stimuli (ES) were applied to myotubes perfused with 500 μM Mn²⁺. Myotubes exposed to 10 μM TCS had negligible fluorescence quench compared with control, suggesting an inhibition of ECCE. (B) Ca²⁺ entry was monitored in ryanodine-treated myotubes loaded with the Ca²⁺ indicator Fluo-4. Ca²⁺ entry was induced by ES. Ryanodine-treated myotubes did not respond to caffeine, hence the rising signal represented pure ECCE, a process completely suppressed by 10 μM TCS. All error bars represent SEM. ***P < 0.001, t test.