Study of heat stress intolerance (HSI)

HSI tests were performed on 6- to 10-month-old (30–50 g) male C57BL/6J WT mice (n = 5), R163C heterozygous mice (n = 6), or T4826I homozygous mice (n = 6). Mice from each genotype were randomly assigned to 2 treatment groups: 0.5% aqueous methyl cellulose vehicle control or 500 mg/kg bw CP (50 mg/ml). At 5 am all mice were dosed by oral gavage (18G x 1.5″, 2 mm flex polytetrafluoroethylene with metal bite protector [Cadence Science, Cranston, Rhod Island]) since p.o. is recognized as the most efficient method of entry for CP (Bentley et al., 2010b). Although absorption of CP is incomplete and dose related, we proceeded with a high-dose oral exposure protocol established by Dupont (FAO/WHO, 2008). A high dose was chosen because: (i) CP does not readily go into solution in aqueous methyl cellulose, and (ii) it permits a small volume of CP by gavage, minimizing potential variability in the actual CP dose administered. Post dosing, the mice were permitted to freely roam in their respective home cages with food and water ad libitum for 12 h (T_max) to achieve C_max (5.1 µg/g) (Bentley et al., 2010b; FAO/WHO, 2008; Himmelstein, 2006a; Wolterink, 2008) to ensure CP broadly distributes to organs, including highly perfused skeletal muscle compartments. At 5 pm, a rectal probe was inserted in each mouse to measure and continuously record core temperature. They were then restrained in medium mouse holders (Kent Scientific Corp., Torrington, Connecticut) to acclimate for 5 min at room temperature before initiating the heat stress protocol in a 38°C incubator. Heat stress was terminated after 60 min or earlier if the mouse underwent a fulminant MH episode and died. Core body temperature for the duration of the study was recorded by the Temperature Controller 324B and CL-100 from Warner Instrument, which was connected with the digitizer from Axon Instruments Digitizer 1320A at a sampling rate of 1 Hz by AxoScope software (V.9.0; Molecular Devices, Sunnyvale, California).