Abstract
Malignant hyperthermia (MH) and central core disease in humans have been associated with mutations in the skeletal ryanodine receptor (RyR1). Heterozygous mice expressing the human MH/central core disease RyR1 R163C mutation exhibit MH when exposed to halothane or heat stress. Considering that many MH symptoms resemble those that could ensue from a mitochondrial dysfunction (e.g. metabolic acidosis and hyperthermia) and that MH-susceptible mice or humans have a higher than normal cytoplasmic Ca(2+) concentration at rest, we evaluated the role of mitochondria in skeletal muscle from R163C compared with wild type mice under basal (untriggered) conditions. R163C skeletal muscle exhibited a significant increase in matrix Ca(2+), increased reactive oxygen species production, lower expression of mitochondrial proteins, and higher mtDNA copy number. These changes, in conjunction with lower myoglobin and glycogen contents, Myh4 and GAPDH transcript levels, GAPDH activity, and lower glucose utilization suggested a switch to a compromised bioenergetic state characterized by both low oxidative phosphorylation and glycolysis. The shift in bioenergetic state was accompanied by a dysregulation of Ca(2+)-responsive signaling pathways regulated by calcineurin and ERK1/2. Chronically elevated resting Ca(2+) in R163C skeletal muscle elicited the maintenance of a fast-twitch fiber program and the development of insulin resistance-like phenotype as part of a metabolic adaptation to the R163C RyR1 mutation.
Highlights
Similar RyR1 Protein Expression in Skeletal Preparations from Wild Type (WT) and R163C Mice—To ensure that the genetically modified mice had the same level of RyR1 protein as WT, protein expression was evaluated by Western blot analysis of whole skeletal muscle membranes using monoclonal antibody 34C that selectively recognizes mouse RyR1 over RyR2 by Western blotting [69]
Considering that a subpopulation of mitochondria has been found closely attached to sarcoplasmic reticulum (SR) [87, 88], and these mitochondria are exposed to higher local resting Ca2ϩ concentrations in skeletal muscle [89, 90], it is likely that our calcium values represent an underestimation of the actual calcium concentrations that could be found in mitochondria closely associated with SR [90]
The values for total calcium contents of WT mitochondria evaluated in this study were within the range of those previously reported for rodent skeletal muscle using ICP-MS or other techniques [82,83,84,85,86]
Summary
Animals—All experiments on animals from creation of MH/CCD mice to establishment of their physiological and biochemical phenotypes were conducted using protocols approved by the institutional animal care and use committees at the Australian National University, Harvard Medical School, and the University of California, Davis, essentially as described previously [29]. Mouse diaphragm was excised and prepared for glucose and oxygen uptake as described previously [58] with the following modifications: 30 – 45 mg of wet weight muscle was placed in the oxygen chamber using the apparatus described above in buffered modified Ringer solution with 10 mM glucose at 22 °C, followed by the sequential addition of the following chemicals separated by 5– 8-min intervals (enough to detect a significant rate): 5 g/ml oligomycin, 5 nM carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and 5 M rotenone; triplicate aliquots were taken at time 0 and at 2.5 h to evaluate glucose and lactate These compounds were determined using an YSI 2300 STAT Plus glucose analyzer (YSI Inc., Yellow Springs, OH). Values with p Յ 0.05 were considered statistically significant
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
