Experimental protocol
With approval of the local animal care committee (Government of Unterfranken, Wuerzburg, Germany, No. 39/13), homozygous MHS and MHN Pietrain pigs were investigated. The pigs were purchased from a local farmer (Farm Lippert, Euerdorf, Germany) and were derived from several long-standing colonies. All the pigs used were related to one another to varying degrees up to first degree. MH susceptibility or wild type was determined by DNA analysis (GeneControl GmbH, Poing, Germany) prior to the investigation. Homozygous cysteine for arginine substitution at position 615 of RYR1 indicating MH susceptibility was proven in nine pigs while eight animals were homozygous negative for this mutation.
After insertion of an intravenous line into an ear vein, general anesthesia was induced by application of fentanyl (0.01 mg/kg) (Janssen, Neuss, Germany) and thiopental (10 mg/kg) (Nycomed, Konstanz, Germany). Afterwards, the trachea was intubated without administration of a muscle relaxant using an orotracheal tube 9.0 mm internal diameter (Teleflex Medical GmbH, Kernen, Germany). The pigs were mechanically ventilated with an inspiratory oxygen fraction of 0.3. Ventilator settings were adjusted to keep an end-tidal PCO2 of 32–39 mmHg (respiratory-rate: 10–16 breaths/min; tidal volume: 8–12 ml/kg; positive end-expiratory pressure: 5 mmHg). Anesthesia was maintained by continuous intravenous administration of thiopental (10 mg/kg/h) and fentanyl (0.01–0.02 mg/kg/h). Radiant heat application and warming blankets were used to ensure normothermia. Vital parameters were monitored continuously by invasive blood pressure monitoring in the tibial artery and by peripheral oxygen saturation, electrocardiography, and rectal temperature measurements.
After induction of general anesthesia, muscle specimens were excised from the left distal biceps femoris muscle, placed in carboxygenated (95% oxygen, 5% carbon dioxide) Krebs-Ringer’s solution (NaCl 118.1 mM; KCl 3.4 mM; CaCl2 2.5 mM; MgSO4 0.8 mM; KH2PO4 1.2 mM; NaHCO3 25.0 mM; Glucose 11.1 mM) (Pharmacy of the University Hospital, Wuerzburg, Germany) and immediately transported to the laboratory. Following preparation of the excised muscle into single muscle strips, length and wet weight of each muscle bundle was measured. For in vitro contracture test (IVCT) investigations, single muscle strips were mounted vertically in the experimental bath perfused with carboxygenated Krebs-Ringer’s solution at 37 °C, fixed to an isometric force transducer (Hugo Sachs, Type 809, March, Germany) and stimulated electrically with a supra-maximal square wave stimulus at 1 ms duration and a frequency of 0.2 Hz (Hugo-Sachs-Elektronik, Type 215/I, March, Germany). Resting tension and twitch of the muscle strips were recorded continuously by a digital recording system (HSE-ACAD Software, Hugo Sachs, March, Germany). Only muscle specimens with initial twitch response of at least 10 mN were included in the investigation. Two different muscle bundles per animal were analyzed. After stable conditions were reached, muscle bundles were incubated successively with increasing concentrations (0.065, 0.125, 0.5, 1.0, 10 and 50 μg/ml) of a commercially available levosimendan preparation (Orion Pharma GmbH, Hamburg, Germany) at 3 min intervals. To ensure sufficient viability of the muscle strips, investigations were performed within 3 h after muscle harvest.
After completion of the experiments, the pigs were euthanized in deep anesthesia by thiopental bolus application (0.1 g/kg).
Statistics
Data are presented as median and interquartile range. A quasi-randomization was given by the availability of the animals independently from the MH diagnosis. Statistical evaluation was performed using D’Agostino & Pearson test for normal distribution and student’s t-test (length and weight of muscle strips, pre-drug resting tension and twitch force, maximum contracture) or 2-way-ANOVA with post-hoc Sidak’s test for multiple comparisons (changes of twitch force, developing contractures) for differences between MHS and MHN groups. P < 0.05 was considered statistically significant.