Chloroform extract of hog barn dust modulates skeletal muscle ryanodine receptor calcium-release channel (RyR1)

Abstract

Skeletal muscle weakness is a reported ailment in individuals working in commercial hog confinement facilities. To date, specific mechanisms responsible for this symptom remain undefined. The purpose of this study was to assess whether hog barn dust (HBD) contains components that are capable of binding to and modulating the activity of type 1 ryanodine receptor Ca²⁺-release channel (RyR1), a key regulator of skeletal muscle function. HBD collected from confinement facilities in Nebraska were extracted with chloroform, filtered, and rotary evaporated to dryness. Residues were resuspended in hexane-chloroform (20:1) and precipitates, referred to as HBD_org, were air-dried and studied further. In competition assays, HBD_org dose-dependently displaced [³H]ryanodine from binding sites on RyR1 with an IC₅₀ of 1.5 ± 0.1 μg/ml (K_i = 0.4 ± 0.0 μg/ml). In single-channel assays using RyR1 reconstituted into a lipid bilayer, HBD_org exhibited three distinct dose-dependent effects: first it increased the open probability of RyR1 by increasing its gating frequency and dwell time in the open state, then it induced a state of reduced conductance (55% of maximum) that was more likely to occur and persist at positive holding potentials, and finally it irreversibly closed RyR1. In differentiated C₂C₁₂ myotubes, addition of HBD triggered a rise in intracellular Ca²⁺ that was blocked by pretreatment with ryanodine. Since persistent activation and/or closure of RyR1 results in skeletal muscle weakness, these new data suggest that HBD is responsible, at least in part, for the muscle ailment reported by hog confinement workers.

pork consumption is increasing worldwide, and to keep up with demand, farmers are continuously expanding the size of their operations (26, 36). In developed and middle-income countries, hogs are housed in large confinement facilities, where they are fed a diet rich in grains and beans in preparation for market. The dust from the feed mixes with feces, urine, dander, bacteria, molds, and pungent volatile substances emanating from nearby manure pits to produce what is referred to as hog barn dust (HBD) (9, 11, 38). A growing body of literature indicates that HBD is an underlying cause for the increased incidence of health-related problems in workers in these facilities, including shortness of breath, wheezing, coughing, chest tightness, and asthma-like syndrome (10, 14, 34, 35, 37, 39, 40). Individuals living in proximity to these confinement facilities also reported increased confusion, depression, tension, and lack of vigor (31, 35).

The vast majority of studies conducted to date have focused on elucidating mechanisms underlying respiratory illnesses caused by HBD. Work by Larsson and coworkers found that short-term exposure (2–5 h) of healthy volunteers to HBD induces bronchial hyperresponsiveness and inflammation (21, 22). Long-term employees of confinement facilities also exhibit a reduction in 1-s forced expiratory volume (18, 32). Mice exposed to HBD were also found to have significant lung inflammation (28), and the latter likely results from activation of protein kinase C and the release inflammatory mediators, such as IL-6, IL-8, and TNF-α (30, 33, 42). Aqueous extract of HBD (HBD_aq) was also found to increase ciliary beating and adhesion of peripheral blood lymphocytes to bronchial epithelial cells (23, 41). HBD_aq also activates toll-like receptor 2 present on lung epithelial cells, implicating the innate immune system as part of the lung response to HBD (1, 29).

In addition to the well-recognized and studied respiratory ailments, ≈25% of hog confinement facility workers also reported having skeletal muscle aches and fatigue (8, 17). While these ailments could be attributed to the physical work involved in these confinement facilities, the likelihood that HBD is a contributing factor has not been explored. Since organic compounds present in HBD could be absorbed via the skin, we reasoned that lipid-soluble (and even amphipathic) components of HBD could be binding to and disrupting the activity of proteins critical for skeletal muscle function. One of these proteins is the ryanodine receptor, the channel through which Ca²⁺ leaves the sarcoplasmic reticulum (SR) to enter the cytoplasm and evoke contraction (13). Type 1 ryanodine receptor (RyR1) is present in skeletal muscles and in the diaphragm, the major muscle regulating breathing (12, 19). Potentiating basal activity of RyR1 reduces SR Ca²⁺ load, and, as a result, the amplitude of evoked Ca²⁺ release is reduced, leading to muscle weakness (7). Similarly, if RyR1 is unable to activate or open in a timely manner, evoked Ca²⁺ release from the SR is compromised, also leading to muscle weakness. Thus the purpose of this study was to assess whether HBD contains compounds that are capable of binding to and modulating the activity of RyR1.

MATERIALS AND METHODS

Materials

[³H]ryanodine (specific activity 87 Ci/mmol) was purchased from GE Life Sciences (Boston, MA). Phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine were obtained from Avanti Polar Lipids (Alabaster, AL). Dialysis membranes were obtained from Spectrum Laboratories (Rancho Dominguez, CA). All other reagents and solvents used were of the highest grade commercially available.

Extraction of HBD

HBD collected 1–2 m from the floor of hog confinement facilities in Nebraska were extracted three times with chloroform (CHCl₃, 5 g in 70 ml for 1 h × 3 extractions). Pooled extracts from each sample were gravity filtered (Whatman no. 1 paper) and rotary evaporated to dryness (HBD_CHCl3). HBD_CHCl3 were then redissolved in hexane-CHCl₃ (20:1, 40 ml), and the resultant precipitates were collected by gravity filtration, air-dried, and labeled HBD_org.

Composition of HBD_org

The composition of HBD_org was determined by spotting on silica gel thin layer plates and chromatographed using varying ratios of CHCl₃-methanol (MeOH) with either triethylamine (TEA) or acetic acid (HAc). Compounds were visualized under 365-nm wavelength UV light. Plates were sprayed with 1% sulphuric acid and heated to assess for non-UV-active compounds.

Preparation of SR Vesicles

The University of Nebraska Medical Center, Institutional Animal Care and Use Committee approved rabbits used for the study, and their short-term housing adhered to the Institute for Laboratory Animal Research Guide for Care and Use of Laboratory Animals (27). Crude SR membrane vesicles (SR vesicles) were prepared from back and hindleg fast-twitch muscles, as described previously (3). Briefly, after induction of anesthesia with methohexital sodium (150 mg/kg) via an ear vein, fast-twitch muscles were removed, homogenized (speed setting 4.5, ProScientific, Oxford, CT), in isolation buffer [0.3 M sucrose, 10 mM imidazole·HCl, 230 μM phenylmethylsulfonyl fluoride (PMSF), 1.1 μM leupeptin, pH 7.4], and centrifuged at 7,500 g_av (average g) for 20 min. The supernatant was discarded, and the pellet was resuspended in fresh isolation buffer, homogenized for 1-s time at speed setting 5.5, and centrifuged at 11,000 g_av for 20 min. The supernatant was then filtered through cheesecloth, and SR vesicles were obtained by sedimentation at 85,000 g_av (Beckman Ultra Centrifuge, Ti45 rotor) for 30 min.

Preparation of RyR1 Reconstituted into Proteoliposomes

Proteoliposomes containing RyR1 were prepared as described earlier by Lai and coworkers (20) with some modifications. SR vesicles were fractionated by placing on top a discontinuous sucrose gradient (26 ml each, 0.8, 1.0, 1.2, and 1.5 M) and centrifuging at 110,000 g_av for 2 h (Ti45 rotor). The vesicles at the interface between 1.2 and 1.5 M sucrose were collected, resuspended in fresh isolation buffer, and harvested by centrifugation at 110,000 g_av for 2 h. The resultant pellet enriched in junctional SR vesicles was then solubilized in buffer (19 ml) containing 1.0 M NaCl, 0.05 mM EGTA, 0.35 mM Ca²⁺, 5 mM AMP, 20 mM Na/PIPES, pH 7.4, 0.3 mM Pefabloc, 0.03 mM leupeptin, 0.9 mM DTT, 1.5% CHAPS, and 5 mg/ml phosphatidylcholine for 10 min at room temperature (24°C), followed by 1 h embedded in ice. CHAPS-insoluble material was removed by centrifugation at 26,163 g_av (Beckman Ultra Centrifuge, Ti75 rotor) for 30 min at 4°C, and the supernatant was further fractionated on a linear sucrose gradient (7–15% sucrose, 34 ml per tube, 6 tubes, 3 ml of solubilized protein per tube) by centrifugation at 89,454 g_av (Beckman Ultra Centrifuge, SW28 rotor) for 17 h at 4°C. [³H]ryanodine (3.3 nM) was added to one of the tubes to serve as a guide for RyR1 purification. After centrifugation, 2.0-ml aliquots from the [³H]ryanodine-labeled tube were collected from the bottom of the tube upwards and counted to determine the location of [³H]ryanodine and RyR1. Twenty-microliter volumes from each aliquot were mixed with gel dissociation medium and electrophoresed on 4–15% linear gradient Tris-glycine polyacrylamide gel at 150 V for 180 min and then silver stained according to manufacturer's protocol (BioRad, Burlingame, CA). RyR1-containing fractions from the five unlabeled tubes were pooled and dialyzed at 4°C for 44 h in buffer containing 0.5 M NaCl, 0.1 mM EGTA, 0.2 mM CaCl₂, 10 mM Na/PIPES, pH 7.4, 0.1 mM DTT, and 0.4 mM PMSF with three buffer changes at 2, 6, and 15 h. After dialysis, proteoliposomes containing RyR1 were diluted 1:1 in buffer containing 0.1 mM EGTA, 0.2 mM CaCl₂, 10 mM Na/PIPES, pH 7.4, 0.1 mM DTT, and 0.4 mM PMSF and centrifuged at 163,520 g_av (Ti75 rotor) for 2 h. The pellet was then resuspended in 0.1 M NaCl, 10 mM Na/PIPES, pH 7.4, and 0.3 M sucrose; aliquoted (100 μl); quick-frozen in liquid nitrogen; and stored frozen in the vapor phase of liquid nitrogen until use.

Competition Binding Assays

Competition binding assays were used to assess the ability of HBD_org to bind to RyR1. This assay was also used to calculate IC₅₀ and K_i of HBD_org, as well as general information on the location(s) of the binding site(s) for HBD_org on RyR1.

Binding of HBD_org to RyR1.

SR vesicles (0.1 mg/ml) were incubated in binding buffer consisting of 500 mM KCl, 20 mM Tris·HCl, 0.4 mM CaCl₂, 0.1 mM EGTA, pH 7.4, 6.7 nM [³H]ryanodine with HBD_org (20, 2.0, and 0.2 μg/ml) for 2 h at 37°C. HBD_CHCl3 (220, 110, and 55 μg/ml), ryanodine (1 μM) and the solvent [CHCl₃/MeOH/dimethylformamide (DMF); 2:1:1] used to dissolve HBD_CHCl3 and HBD_org were also used. At the end of the incubation, samples were rapidly filtered through GF/C filters using a cell harvester (Brandel, Gaithersburg, MD) and washed three times with 3 ml of ice-cold binding buffer, and the amount of [³H]ryanodine bound to the filters was determined by liquid scintillation counting (16).

Determination of IC₅₀ and K_i of HBD_org.

For this, 12 concentrations of HBD_org, ranging from 0.2 to 20 μg/ml were used in competition assays. For comparison, the displacement-binding curve for the RyR1-selective ligand ryanodine was run concurrently in each assay. Displacement data were fitted using nonlinear regression to a one- and a two-site competition model given by equations as described by Motulsky and Christopoulos (25) respectively.

$$Y=Y_{\min }+\left(Y_{\max }-Y_{\min }\right)/\left[1+{10}^{\left(x-\log {\text{EC}}_{50}\right)}\right]$$ (1)

$$Y=Y_{\min }+\left(Y_{\max }-Y_{\min }\right)X\left(\text{fraction}1/\left\{1+{10}^{\left[x-\log {\text{EC}}_{50}\left(\text{fraction}1\right)\right]}\right\}+\text{fraction}2/\left\{1+{10}^{\left[x-\log {\text{EC}}_{50}\left(\text{fraction}1\right)\right]}\right\}\right)$$ (2)

where Y is specific binding, X is the logarithm of the amount of the unlabeled ligand, and log IC₅₀ is the competitor concentration that displaces one-half of the specifically bound [³H]ryanodine. Goodness of fit was evaluated by F-test.

Equilibrium inhibition constant (K_i) for HBD_org was calculated using the Cheng-Prusoff relationship (6) given by equation 3

$$K_{\text{i}}={\text{IC}}_{50}/\left[1+\left(L/K_L\right)\right]$$ (3)

where L is the concentration of [³H]ryanodine used (6.7 nM) and K_L is the equilibrium dissociation constant of [³H]ryanodine (2.4 nM for RyR1) (4).

Ca²⁺-dependency of HBD_org actions.

Ca²⁺ is routinely used in the binding buffer to activate or open RyR1, and, under these conditions, the amount of [³H]ryanodine bound to RyR1 is substantially increased. In the absence of Ca²⁺ or in high Ca²⁺ (>900 μM), RyR1 becomes deactivated or closed, and the amount of [³H]ryanodine bound to RyR1 decreases precipitously (2). We, therefore, reasoned that competition assays employing a fixed amount of HBD_org in the presence of varying amounts of Ca²⁺ could provide insights into whether the actions of HBD_org are dependent on the open or closed conformation of RyR1. SR membrane vesicles (0.1 mg/ml) were incubated in binding buffer containing 2.0 μg/ml HBD_org and increasing amounts of Ca²⁺ (2–3,900 μM) for 2 h at 37°C. After incubation, vesicles were filtered and washed, and the amount of [³H]ryanodine bound was determined using liquid scintillation counting.

Single-channel Measurement and Analyses

Single-channel analyses were performed as described earlier (5). Briefly, phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine in a ratio of 5:3:2 (35 mg/ml of lipid) in n-decane were painted across a 200-μm-diameter hole of the bilayer cup. Purified RyR1 in proteoliposomes was then fused to the bilayer. The side of the bilayer to which the proteoliposomes were added was designated the cis side. The trans side was defined as ground. Single channels were recorded in symmetric KCl buffer solution (0.25 mM KCl, 20 mM K/HEPES, pH 7.4) with 3.3 μM Ca²⁺ or as specified. In this study, free calcium concentrations were titrated against EGTA. HBD_org was made up in CHCl₃/MeOH/DMF (2:1:1) at concentrations up to 500 times higher than that anticipated for use in the bilayer bath (≤1% solvent in bath). All experiments were carried out at room temperature (23–25°C), and HBD_org was added only to the cis chamber. Electrical signals were filtered at 2 kHz, digitized at 10 kHz, and analyzed as described earlier (5). Normal gating mode is defined to include periods of rapid channel transitions between the closed and full open states, and subconductance mode is defined as periods in which the channel exhibits a persistently reduced conductance. The magnitude of the subconductance state was calculated by dividing the amplitude of the subconductance induced by the HBD_org by the conductance of the channel before HBD_org (full conductance). Amplitudes were monitored by manual positioning of cursors at each level, corresponding to closed, subconductance, and open-conductance states. Data acquisition and analyses were performed using commercially available instruments and software (Axopatch 1D, Digidata 1200A or 1322A and pClamp 10.0, Axon Instruments, Burlingame, CA). Some data analyses were also carried out using Sigma Plot 10.0 (Stystat Software, Chicago, IL).

Cell Culture and Ca²⁺ Transients

Undifferentiated C₂C₁₂ cells (mouse myoblast cell line), obtained as a gift from Dr. Gregory S. Taylor (Department of Biochemistry, UNMC), were grown in Dulbecco's modified Eagle's medium containing 1.8 mM CaCl₂ (DMEM; Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml gentamicin, pH 7.3) to 70–80% confluency. Cells were then subcultured and plated onto laminin-coated glass-bottom chambers and placed in an incubator for 24 h with 5% CO₂/95% air at 37°C. After this time, the media was changed, fetal bovine serum was reduced to 2%, and cells were allowed to differentiate for 96 h into myotubes. Differentiated myotubes in DMEM (containing 1.8 mM Ca²⁺) were then loaded with fluo 3-AM (5 μM) for 30 min at 37°C, washed, and placed on the stage of a laser confocal microscope (Zeiss Confocal LSM 410 confocal microscope equipped with an Argon-Krypton Laser, 25 mW argon laser, 2% intensity, Thornwood, NJ). HBD_org in dimethylsulfoxide was then manually added to a corner of the glass chamber and allowed to diffuse into the cell medium, and Ca²⁺ transients (ΔF) were recorded. The final dimethylsulfoxide in the chamber was <5%. In some experiments, after loading fluo 3 and washing, 500 mM EGTA were added to DMEM media to chelate extracellular Ca²⁺. In other experiments, differentiated C₂C₁₂ cells were incubated with 50 μM ryanodine 20 min at 37°C before loading with fluo 3-AM. Cells were then challenged with HBD_org, and Ca²⁺ transients were recorded. Fluo 3 was excited by light at 488 nm, and fluorescence was measured at wavelengths of >515 nm. Data were analyzed using Microsoft Excel (Microsoft, Seattle, WA) and Sigma Plot (Systat, Chicago, IL).

Statistical Analyses

Data shown are means ± SE. Differences among concentrations were analyzed using analysis of variance, followed by the Bonferroni (post hoc) test using Prism 4 (GraphPad Software, San Diego, CA).

RESULTS

Preparation of Chloroform Extract of HBD

Chloroform extraction of HBD (HBD_CHCL3) afforded a pale yellow paste (≈5% yield) that exhibited greenish-blue fluorescence under 365-nm wavelength light. At 305 nm, the greenish-blue fluorescence was less intense. When sprayed with 1% sulfuric acid and heated, the major compounds overlaid with the UV-active compounds. Sulfuric acid also revealed low levels of other non-UV active compounds. When redissolved in hexane-CHCl₃ (20:1), a brownish precipitate (HBD_org, 40% yield) was obtained that fluoresced under UV light. The discarded hexane-CHCl₃ (20:1) fraction contained very weak UV-active spots that did not react with sulfuric acid.

Chromatographic Characterization of HBD_org

Basic (TEA) and acidic (HAc) mobile phases of increasing polarities were employed to gain insights into the composition of HBD. When spotted on a thin-layer chromatographic plate and developed using a mobile phase consisting of CHCl₃/MeOH/TEA (97:3:0.05), all four HBD_org extracts contained three major compounds that migrated with R_f 0.65, (#1), 0.52 (#2), 0.52 (#3; Fig. 1A, left, arrows). Compounds 2 and 3 were better resolved in sample 1. In this solvent system, all four HBD_org samples exhibited streaking near the origin of the plate. Increasing the polarity of the basic mobile phase to 7% methanol resulted in the appearance of two additional compounds with R_f values of 0.2 (#4) and 0.15 (#5). Increasing the polarity of the mobile phase to 15% methanol did not reveal any additional compounds. However, the resolution of compounds 4 and 5 was enhanced. In all three basic mobile phases of increasing polarities, bright streaks were seen at the origin, suggesting that HBD_org also contains very polar, nonbasic compounds.

Fig. 1.

Chromatographic mobilities of hog barn dust (HBD) chloroform extract (HBD_CHCl3) precipitates (HBD_org). A and B: 5 μl from 5 mg/ml solutions of HBD_org spotted on a thin-layer silica gel plate and developed for 45 min in solvents containing mixtures of CHCl₃/MEOH with either triethylamine (A) or acetic acid (B). The plates were air-dried, visualized under 365-nm wavelength light, and photographed. R_f values were determined by measuring the distance the compound moved from the origin relative to the distance moved by the solvent. C: 10 μl from 5 mg/ml solution of HBD_org and 10 μl from 1 g/10 ml solution of HBD aqueous extract (HBD_aq) spotted on a thin-layer silica gel plate alongside ryanodine (10 μl from 10 mg/ml).

Replacing TEA with HAc in the mobile phase also revealed the presence of five major compounds in HBD_org (Fig. 1B). Bright streaks were also seen at origin in acid mobile phases. At this time, it is not clear whether the compounds that separated with the basic mobile phases are the same as those that separated with the acidic mobile phases.

Assessing the Ability of HBD_org to Bind to RyR1

HBD_org dose-dependently displaced [³H]ryanodine from RyR1 and, as expected, was ≈10× more potent than HBD_CHCl3 (Fig. 2). Ryanodine (1 μM) displaced >90% of [³H]ryanodine from RyR1. CHCl₃/MeOH/DMF (2:1:1) used to dissolved HBD_CHCl3 and HBD_org (4%) had minimal effect on the binding of [³H]ryanodine to RyR1. HBD_aq did not significantly affect the binding of [³H]ryanodine RyR1 at the concentrations used (20 and 40 μl from a 1 g/10 ml extract in 400 μl of binding buffer).

Fig. 2.

Screening HBD for type 1 ryanodine receptor Ca²⁺-release channel (RyR1) activity. [³H]ryanodine competition assays were conducted with various amounts of HBD_CHCl3, HBD_org, HBD_aq, and solvent controls. For this, sarcoplasmic reticulum (SR) vesicles (0.1 mg/ml) were incubated in binding buffer consisting of 500 mM KCl, 20 mM Tris·HCl, 0.3 mM CaCl₂, 0.1 mM EGTA, pH 7.4, 6.7 nM [³H]ryanodine with HBD_CHCl3 (220, 110, and 55 μg/ml), HBD_org (20, 2.0, and 0.2 μg/ml), ryanodine (1 μM), and the solvent [CHCl₃/MeOH/dimethylformamide (DMF); 2:1:1], for 2 h at 37°C. At the end of the incubation, samples were rapidly filtered through GF/C filters using a cell harvester (Brandel, Gaithersburg, MD) and washed three times with 3 ml of ice-cold binding buffer, and the amount of [³H]ryanodine bound to the filters was determined by liquid scintillation counting. Data for each compound represent means ± SE from four experiments. ^A Dose response for chloroform extract of HBD. ^B Dose response for organic extract of HBD. *Significantly different from control (P < 0.05). #Significantly less than that of 2 and 0.2 μg/ml HBD_org (P < 0.05).

The affinity of HBD_org for RyR1 was then determined using a wide concentration range of HBD_org in displacement assays. As shown in Fig. 3, HBD_org displaced [³H]ryanodine from RyR1 in a concentration-dependent manner. The experimental data fitted well to a one-site binding model (r² = 0.95, F = 0.92), affording an IC₅₀ value of 1.5 ± 0.10 μg/ml. The data also fitted well to a two-site model (r² = 0.95, F = 0.92). Since displacement occurred over two log units, the less complicated single-site model was used for fitting data (25). For comparison, the displacement isotherm of ryanodine is shown in the solid circles, exhibiting an IC₅₀ of 3.8 ± 0.2 ng/ml (IC₅₀ = 7.7 ± 0.4 nM, given molecular mass of 493 Da). Using Eq. 3 with L = 13.59 ng/ml (6.7 nM [³H]ryanodine) and K_L of ryanodine of 4.8 ng/ml (2.4 nM), the K_i for HBD_org binding to RyR1 was calculated to be 0.4 μg/ml. The binding isotherms of RyR1 and HBD_org were also parallel, suggesting that both ryanodine and the active compound(s) of HBD_org are binding to the same site(s) on RyR1.

Fig. 3.

Determining the affinity of HBD_org for RyR1. Displacement [³H]ryanodine binding assays were used to determine the affinity of HBD_org for RyR1. For this, SR vesicles (0.1 mg/ml) were incubated in binding buffer consisting of 500 mM KCl, 20 mM Tris·HCl, 0.3 mM CaCl₂, 0.1 mM EGTA, pH 7.4, and 6.7 nM [³H]ryanodine with 12 concentrations of HBD_org (0.007–15.75 μg/ml) for 2 h at 37°C. At the end of the incubation, samples were rapidly filtered through GF/C filters using a cell harvester (Brandel) and washed three times with 3 ml of ice-cold binding buffer, and the amount of [³H]ryanodine bound to the filters was determined by liquid scintillation counting. Data for each compound represent means ± SE from four experiments. For comparison, ryanodine was also used in this assay.

To gain insight into whether the effect of HBD_org is dependent on the open or closed conformation of RyR1, displacement assays were conducted with varying Ca²⁺ concentrations. As shown in Fig. 4, in the absence of Ca²⁺ or at high Ca²⁺ (≥900 μM), HBD_org was to unable displace [³H]ryanodine from RyR1. However, HBD_org competed with [³H]ryanodine at all concentrations of Ca²⁺ that activated or opened RyR1 (2–300 μM).

Fig. 4.

Determining the Ca²⁺ dependency of HBD_org effect on RyR1. For this, SR vesicles (0.1 mg/ml) were incubated in binding buffer consisting of 500 mM KCl, 20 mM Tris·HCl, 0.1 mM EGTA, pH 7.4, 6.7 nM [³H]ryanodine, with 2.0 μg/ml HBD_org and increasing amounts of Ca²⁺ (2–3,900 μM) for 2 h at 37°C. At the end of the incubation, samples were rapidly filtered through GF/C filters using a cell harvester (Brandel) and washed three times with 3 ml of ice-cold binding buffer, and the amount of [³H]ryanodine bound to the filters was determined by liquid scintillation counting. Data for each compound represent means ± SE from four experiments. *Significantly different from [³H]ryanodine bound in absence of HBD_org (P < 0.05).

Effects of HBD_org on Single RyR1 Channels

Figure 5 shows the elution profile and a silver-stained gel of aliquots collected following solubilization and centrifugation of junctional SR membranes. Fractions 3, 4, and 5 were collected, dialyzed, and used in single-channel assays, to gain additional insight into mechanisms by which HBD_org alters the activity of RyR1. Three distinct effects were observed when HBD_org was added to the cis chamber of the bilayer cup. At a concentration of 3.5 μg/ml, HBD_org increased the probability of RyR1 opening (P_o) from 0.08 ± 0.01 to 0.37 ± 0.05 at +40 mV, and from 0.02 ± 0.01 to 0.35 ± 0.08 at −40 mV within 3 min after addition (Fig. 6, left, 1st and 2nd panels). This increase in P_o resulted from increases in both the frequency of transitions from the closed to the full open state (number of events increased from 556 ± 48 to 841 ± 154/s, P < 0.05) and dwell time in the open state (from 0.39 ± 0.11 to 0.73 ± 0.13 ms, P < 0.05). Dwell time in the closed state also decreased from 4.2 ± 0.8 to 1.2 ± 0.5 ms (P < 0.05). The increase in P_o was seen at both +40 mV and −40 mV holding potentials. The solvent used to dissolve HBD_org had no significant effect on the activity of RyR1 (data not shown).

Fig. 5.

Purification of RyR1 using linear sucrose gradient centrifugation. Sucrose gradient (linear) centrifugation was used to purify RyR1. For this, junctional membrane vesicles were solubilized using CHAPS and then loaded on top of a linear sucrose gradient (7–15% sucrose, 34 ml per tube, ×6 tubes) and centrifuged at 89,454 g_av (average g) for 17 h at 4°C. [³H]ryanodine (3.3 nM) was added to one of the tubes to serve as a guide for RyR1 purification. After centrifugation, 2.0-ml aliquots from the [³H]ryanodine-labeled tube were collected from the bottom of the tube upwards (A) and counted to determine the location of [³H]ryanodine and RyR1 (B). C: 20-μl volumes from each aliquot were mixed with gel dissociation medium also electrophoresed on 4–15% linear gradient Tris-glycine polyacrylamide gel at 150 V for 180 min and then silver stained, according to manufacturer's protocol (BioRad, Burlingame CA). JSRV, junctional SR vesicles; STD, standard.

Fig. 6.

Effects of HBD_org on single RyR1. Single-channel currents were recorded at +40 mV (left, upward deflections) and −40 mV (right, downward deflections) in symmetric KCl buffer solution (0.25 mM KCl, 20 mM K/HEPES, pH 7.4), as described in the text. The top right and left panels show representative recordings of RyR1 in the presence of 3.3 μM cytosolic Ca²⁺ before addition of HBD_org to the cis chamber. The panels below thereafter represent the effect of varying concentrations of HBD_org. The second and third panels from the top show increases in open probability (P_o), the fourth panel shows the induction of a subconductance state, and the fifth and sixth panels show induction of the closed state of the channel. Data shown are representative of six separate channels. HP, holding potential; O, open; C, closed; M, modified.

Tripling the cis concentration of HBD_org to 10.5 μg/ml further increased the P_o of RyR1 at both positive and negative holding potentials. This increase in P_o also resulted from increases both in its gating frequency of RyR1 (number of events increased to 748 ± 201, P < 0.05) and its dwell time in the open state (1.8 ± 0.9 ms). Dwell time in the closed state also decreased to 0.6 ± 0.2 ms. Increasing the amount of HBD_org in the cis chamber of the bilayer bath to 17.5 μg/ml induced a state of reduced conductance (subconductance) with current amplitude of 55 ± 4% of the fully opened state (391 ± 45 vs. 710 ± 90 pS, P < 0.05). Induction of this subconductance state (with P_o = 1) was more likely to occur and persisted for longer times at +40 than at −40 mV. In fact, the time to modification and the probability of occurrence of the subconductance state were about five times more likely at positive holding potentials than at negative holding potentials. At positive holding potentials and with 17.5 μg/ml HBD_org, no transitions were observed from the subconductance state to either the full open or closed states (Fig. 6, left, 4th panel). At −40 mV, the subconductance state was noticeably noisier than at positive holding potentials, and the channel transitioned to the fully open state with high P_o. Increasing the concentration of HBD_org even further to 21.5 μg/ml resulted in the channel predominantly residing in a subconductance state at both positive and negative holding potentials, with occasional transitions to the closed state. Higher concentrations (≥35.0 μg/ml) of HBD_org resulted in irreversible channel closure. Figure 7 shows the pooled data of the effects of HBD_org on the P_o of RyR1.

Fig. 7.

Effects of HBD_org on the P_o of RyR1. The currents of single RyR1 incorporated into lipid bilayers were recorded at +40 mV and −40 mV (in symmetric KCl buffer), and P_o of the channel (transition from closed to full open state) in the presence of varying amounts of HBD_CHCl3 in the cis chamber were plotted. Data shown are means ± SE from six channels.

The effects of HBD_org on gating and conductance of RyR1 are reminiscent of that seen with the plant alkaloid ryanodine. Accordingly, we assessed whether ryanodine might be present in HBD_org. When spotted on a silica gel thin-layer chromatographic plate and developed using a mobile phase consisting of CHCl₃/MeOH/TEA (88:12:0.05), neither HBD_org nor HBD_aq contained any compound with a chromatographic mobility (R_f) similar to that of ryanodine (Fig. 1C, arrow, R_f for ryanodine = 0.39).

HBD_org Triggers Ca²⁺ Release From Differentiated C₂C₁₂ Myotubes

Binding and single-channel studies indicate that HBD_org contains compound(s) that binds to and modulates the activity of RyR1. We then tested whether HBD_org can also modulate the activity of RyR1 in intact, differentiated C₂C₁₂ myotubes (skeletal muscle cells). When added to fluo 3-loaded C₂C₁₂ myotubes, HBD_org (50 μg/ml) increased cytoplasmic Ca²⁺ (Fig. 8A, ii). The peak rise in Ca²⁺ (ΔF) was 1.9 ± 0.2-fold over basal and occurred ≈80 s after addition of HBD_org. The transient decayed to basal 300 s after the addition of HBD. While caffeine (10 mM) was able to trigger Ca²⁺ release in differentiated C₂C₁₂ cells (Fig. 8A, i), it was unable to do so after addition of 50 μg/ml HBD_org (Fig. 8A, iii). Addition of 250 μg/ml HBD_org induced a more robust increase in intracellular Ca²⁺ (Fig. 8A, iv). The peak rise in fluorescence (ΔF) was 3.6 ± 0.2-fold over basal and occurred ≈80 s after the addition of HBD_org. Treating differentiated C₂C₁₂ cells with 50 μM ryanodine for 20 min to block intracellular RyR1 blunted the increase in cytoplasmic Ca²⁺ induced by HBD_org (Fig. 8, Av and B). Using EGTA to chelate extracellular Ca²⁺ minimally impact the ability of HBD_org to trigger Ca²⁺ release from C₂C₁₂ cells, indicating that HBD_org is inducing intracellular Ca²⁺ release (data not shown).

Fig. 8.

Ability of HBD_org to trigger Ca²⁺ release from intracellular stores. Differentiated C₂C₁₂ myotubes grown on laminin-coated glass bottom chambers were loaded with fluo 3-AM (5 μM) for 30 min at 37°C, washed, and placed on the stage of a laser confocal microscope (Zeiss Confocal LSM 410 confocal microscope equipped with an Argon-Krypton Laser, 25 mW argon laser, 2% intensity, Thornwood, NJ). A: caffeine (10 mM; i) and HBD_org (50 μg/ml; ii, and 250 μg/ml; iv) were added to the chambers, and Ca²⁺ transients recorded. iii: Three minutes after addition of 50 μg/ml HBD_org and Ca²⁺ transient generation, caffeine (10 mM) was added. v: In separate experiments, differentiated C₂C₁₂ cells were incubated with 50 μM ryanodine 20 min at 37°C before loading with fluo 3-AM and exposure to 250 μg/ml HBD_org. B: fold increase in cytoplasmic Ca²⁺ following addition of HBD_org (50 and 250 μg/ml, with and without ryanodine). Values are means ± SE of peak Ca²⁺ release for >10 cells from 4 separate experiments. *Significantly different from 50 μg/ml HBD_org (P < 0.05). **Significantly different from 50 μg/ml HBD_org and 250 μg/ml HBD_org (P < 0.05).

DISCUSSION

Hog production is a major agricultural industry in the United States, Europe, and several middle-income countries (36). While the economic impact is clear, environmental and health-related issues resulting from this industry are experiencing an avalanche of public discussion. Leading the debate is the issue of the effect of confinement dust on the health and well-being of workers and individuals living in communities surrounding these facilities. It is becoming clearer that inhalation of HBD contributes to the increased incidence of respiratory ailments, including shortness of breath, wheezing, coughing, and chest tightness, among confinement facility workers (10, 14, 34, 37, 39, 40). However, what is not clear whether HBD is also contributing to skeletal muscle weakness and fatigue, and, if it does, what are the mechanism(s) (8, 17)?

The principal finding of the present study is that chloroform extracts of HBD collected from confinement facilities across Nebraska bind to and modulate the activity of RyR1. Using displacement assays, we found that all four samples of HBD_org exhibited high affinities for RyR1 (K_i = 0.39 μg/ml) and that the binding of HBD_org to RyR1 is preferred when the channel is in an activated or opened state. The latter is very important, as it suggests that active or working muscles are more prone to the effects of HBD than nonactive muscles. Using black lipid bilayers, we also found that HBD_org exhibited three distinct, dose-dependent effects on binding to RyR1. At low microgram amounts, HBD_org increased the P_o of RyR1 by mechanisms that involved increasing both the gating of RyR1 and its dwell time in the open state. At higher concentrations of HBD_org, RyR1 transitioned into a state of reduced conductance (55% of full open) with a P_o of 1. In the presence of even more HBD_org, RyR1 irreversibly closed. Irreversible closure of RyR1 could be a result of slow dissociation of HBD_org from RyR1. Thus HBD_org is a potent modulator of RyR1.

Parallel findings were also observed in skeletal muscle myotubes. When added to the culture chamber, HBD_org increased Ca²⁺ in differentiated C₂C₁₂ myotubes in a dose-dependent manner. The rise in intracellular Ca²⁺ persisted for ∼1 min and then decayed to near or even below basal. Removal of Ca²⁺ from the medium minimally impacted the ability of HBD_org to increase intracellular Ca²⁺, indicating that the Ca²⁺ rise is originating from inside and not from outside the cell. Pretreating differentiated C₂C₁₂ cells with ryanodine to close intracellular RyR1 blunted the ability of HBD_org, suggesting that the initial intracellular Ca²⁺ rise occurs via activation of RyR1. The inability of caffeine to trigger Ca²⁺ release post-HBD_org treatment is also consistent with the initial intracellular Ca²⁺ rise occurring via activation of RyR1. From single-channel studies, the rise in intracellular Ca²⁺ in C₂C₁₂ cells is likely the result of HBD_org increasing the gating frequency and inducing a state of reduced conductance of RyR1. The lower basal cytoplasmic Ca²⁺ post-HBD_org treatment and the inability of caffeine to trigger Ca²⁺ release post-HBD_org suggest that HBD_org is shutting RyR1, consistent with single-channel data.

Increasing the basal activity of RyR1, as is the case when its P_o is increased or a subconductance state is induced, would result in leakage of Ca²⁺ from the SR. As a result, the amount of Ca²⁺ available for contraction following depolarization would be compromised. Inactivating or closing RyR1, as would be the case with higher amounts of HBD_org, would result in excitation-contraction uncoupling, as little or no Ca²⁺ would be released from the SR via an inactivated RyR1. Since effective skeletal muscle contraction requires the coordinated release of sufficient amounts of Ca²⁺ from the SR, these data provide, for the first time, a physiological rationale for the muscle weakness reported by hog confinement facility workers (8, 17). Since RyR1 is also a key Ca²⁺ release channel involved in diaphragm contraction (12, 19), the data from the present study also suggest that HBD could be compromising breathing, by another mechanism, i.e., impairment in diaphragm function. However, more work is needed to establish this. It would also be of interest to assess whether HBD_org is capable of modulating the activity of other cells that expresses RyR1 (13, 15).

HBD_org could enter into the circulation and access RyR1 via two major routes: absorption though the skin, and inhalation through the lung. While most companies require individuals working in concentrated animal feeding operations to wear protective coverings over hair and clothing and to use a respirator (24), it is conceivable that necks and faces are less likely to be protected, and these areas could serve as absorption sites for HBD components. Sweat produced during the course of work could also serve to increase the amount of HBD accumulated in these areas. The acidity of sweat is not likely to affect the components of HBD, as we did not observe any degradation in our chromatographic studies (Fig. 1). To what extent workers comply with the clothing policy is not clear. However, it is conceivable that thick or low porosity clothing worn during hot summer days could be a hindrance for workers.

Individuals working in concentrated animal feeding operations are also required to wear respirators. However, confinement facility workers find respirators to be a nuisance as they can become easily clogged (24). If these masks are not personalized, as is likely the case, leakage will occur, and inhalation could also serve to increase circulating concentrations of HBD components. However, the tolerance to inhalation is likely to be less than that which can be attached to the skin.

A surprising finding of the present study is that HBD_org exhibits actions on RyR1 similar to those seen with the plant alkaloid ryanodine (5, 13, 20). Specifically, at low concentrations, it activated or increased the P_o of RyR1, and at higher concentrations it closed RyR1. However, none of the HBD samples analyzed to date contained any compounds that migrated with a chromatographic mobility similar to that of ryanodine (or its congener 9,21-didehydroryanodine) (Fig. 1C). These data are exciting, as they indicate another source for ligands that can modulate the activity of RyR1. Which component of HBD_org produces ryanodine-like actions is not known at this time. Because of this unexpected finding, it would be of great interest to assess whether HBD_org can also modulate the activities of the other isoforms of RyR, namely type 2 ryanodine receptor (RyR2, predominant in heart) and type 3 ryanodine receptor (RyR3, predominant in diaphragm and brain).

It should be mentioned that, during preliminary fractionation of HBD_org, a white crystalline solid was isolated and identified as lauric acid by ¹H- and ¹³C-nuclear magnetic resonance spectroscopy (data not shown). In between batches of animals, confinement facilities are hosed and cleaned with commercial detergents, and it is likely that residual detergent molecules are serving as nuclei to attract HBD particles. In preliminary studies, up to 10 μg/ml lauric acid had no effect on the ability of [³H]ryanodine to bind to RyR1 (data not shown).

In conclusion, we have shown 1) using rabbit skeletal muscle membrane vesicles that HBD_org displaces [³H]ryanodine from RyR1; 2) using purified RyR1 reconstituted into lipid bilayers that HBD_org modulates the gating and conductance of RyR1; and 3) using differentiated C₂C₁₂ that HBD_org triggers release of Ca²⁺ from intracellular stores via activation of RyR1. These new data provides a mechanistic rationale for the skeletal muscle weakness reported by hog confinement facility workers. They also raise several intriguing questions. Since RyR1 is present in many other cell types (the diaphragm, monocytes, B-cells, T-cells, dendritic cells, neurons, etc.), is modulation of RyR1 central to the reported effects of HBD? Also, since the actions of HBD on RyR1 are similar to that of the plant alkaloid ryanodine, could HBD also interact with and alter the activities of the other ryanodine receptor isoforms, i.e., RyR2 and RyR3? If the latter holds, then HBD could be the underlying cause for additional ailments. Identify the components of HBD that bind to and modulate the activity of RyR1 is, therefore, of utmost importance.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).