Dust From Pig Confinement Facilities Bind to and Activate Skeletal Muscle Ryanodine Receptor Calcium-Release Channel (RyR1)

Abstract

Individuals working in industrial pig farms and residents in communities surrounding these confinement facilities report increased muscle weakness and fatigue. However, causative factors and molecular mechanisms responsible for this remain poorly characterized. In this study we investigated whether dust from confinement facilities contains components that can bind to and activate RyR1. Dust collected 1–2 meters from the ground of pig confinement facilities in Nebraska were extracted with chloroform, filtered and rotor evaporated to dryness. The residues were redissolved in hexane:chloroform (20:1) and precipitates herein referred to as HEX-INS were filtered and air-dried. Thin layer chromatography revealed three major compounds in HEX-INS fractions and 1H NMR suggests these compounds contained fatty acid moieties. In binding assays, HEX-INS displaced [3H]ryanodine from rabbit skeletal muscle RyR1 in a dose-dependent manner, with an IC50 of 2.0μg/ml. HEX-INS also displaced [3H]ryanodine from RyR2 (dog heart), but was significantly less potent (IC50 of 21.0μg/ml). HEX-INS displacement curves were parallel to that of ryanodine and occurred principally at activating Ca2+ concentrations. In single channel studies, HEX-INS increased open probability of RyR1 in a dose-dependent manner (3.5 to 14.0μg/ml, cis Ca2+ 3μM). At 17.5μg/ml, HEX-INS induced RyR1 in a state of reduced conductance (55.2% of maximum). This subconductance state was more likely to occur and persist at positive holding potentials. Increasing HEX-INS further to 21.0μg/ml resulted in reversible channel closure. At a higher cis Ca2+ (20μM), lower amounts of HEX-INS were required for induction of the subconductance state and channel closure. These data are the first to demonstrate that pig confinement dust contains component(s) that selectively bind to and activate RyR1, providing a mechanism rationale for reported muscle weakness and fatigue. (Supported in part by grants from NIH to DJR and KRB)