Basal Bioenergetic Abnormalities in Skeletal Muscle from Ryanodine Receptor Malignant Hyperthermia-susceptible R163C Knock-in Mice

Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2010; 286(1):99-113. DOI: 10.1074/jbc.M110.153247
Source: PubMed


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.

Download full-text


Available from: Eleonora Napoli,
32 Reads
  • Source
    • "All mice utilized in this study were genotyped for the wild-type or mutated NNT as described [39]. Genomic DNA was isolated from 5 mg of hindbrain as previously described [56]. Concentration and purity of DNA was measured at an absorbance of 260 nm and 280 nm on a Tecan infinite M200 Nanoquant (Tecan, Austria). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Familial Glucocorticoid Deficiency (FGD) is a rare autosomal recessive disorder that is characterized by isolated glucocorticoid deficiency. Recently, mutations in the gene encoding for the mitochondrial nicotinamide nucleotide transhydrogenase (NNT) have been identified as a causative gene for FGD; however, no NNT activities have been reported in FGD patients carrying NNT mutations.Methods Clinical, biochemical and molecular analyses of lymphocytes from FDG homozygous and heterozygous carriers for the F215S NNT mutationResultsIn this study, we described an FGD-affected Japanese patient carrying a novel NNT homozygous mutation (c.644 T > C; F215S) with a significant loss-of-function (NNT activity = 31% of healthy controls) in peripheral blood cells' mitochondria. The NNT activities of the parents, heterozygous for the mutation, were 61% of controls.Conclusions Our results indicated that (i) mitochondrial biogenesis (citrate synthase activity) and/or mtDNA replication (mtDNA copy number) were affected at ≤ 60% NNT activity because these parameters were affected in individuals carrying either one or both mutated alleles; and (ii) other outcomes (mtDNA deletions, protein tyrosine nitration, OXPHOS capacity) were affected at ≤ 30% NNT activity as also observed in murine cerebellar mitochondria from C57BL/6 J (NNT-/-) vs. C57BL/6JN (NNT+/+) substrains.General significanceBy studying a family affected with a novel point mutation in the NNT gene, a gene-dose response was found for various mitochondrial outcomes providing for novel insights into the role of NNT in the maintenance of mtDNA integrity beyond that described for preventing oxidative stress.KeywordsFamilial glucocorticoid deficiencymitochondrial biogenesismitochondrial replicationnicotinamide nucleotide transhydrogenaseoxidative phosphorylationoxidative stress
    Biochimica et Biophysica Acta - Clinical 06/2015; 3. DOI:10.1016/j.bbacli.2014.12.003
  • Source
    • "Neutrophils were spun at 4,000 rpm for 4 min (MSE microcentaur) prior to resuspension in lysis buffer (20 mM MOPS, 50 mM NaF, 50 mM β-glycerophosphate, 50 mM Na3VO4, 1 % Triton X-100, 1 mM DTT, 1 mM AEBSF and 1 % protease [27] and phosphatase inhibitor cocktails [28]). Lysis of neutrophils was performed on ice for 30 min with occasional vortexing. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Neutrophils are abundant, short-lived leukocytes that play a key role in the immune defense against microbial infections. These cells die by apoptosis following activation and uptake of microbes and will also enter apoptosis spontaneously at the end of their lifespan if they do not encounter a pathogen. Adiponectin exerts anti-inflammatory effects on neutrophil antimicrobial functions, but whether this abundant adipokine influences neutrophil apoptosis is unknown. Here we report that adiponectin in the physiological range (1-10 μg/ml) reduced apoptosis in resting neutrophils, decreasing caspase-3 cleavage and maintaining Mcl-1 expression by stabilizing this anti-apoptotic protein. We show that adiponectin induced phosphorylation of AMP-activated kinase (AMPK), protein kinase B (PKB), extracellular signal-regulated kinase (ERK 1/2) and p38 mitogen activated protein kinase (MAPK). Pharmacological inhibition of AMPK, PKB and ERK 1/2 ablated the pro-survival effects of adiponectin and treatment of neutrophils with an AMPK specific activator (AICAR) and AMPK inhibitor (compound C) respectively decreased and increased apoptosis. Finally, activation of AMPK by AICAR or adiponectin also decreased ceramide accumulation in the neutrophil cell membrane, a process involved in the early stages of spontaneous apoptosis, giving another possible mechanism downstream of AMPK activation for the inhibition of neutrophil apoptosis.
    Apoptosis 08/2013; 18(12). DOI:10.1007/s10495-013-0893-8 · 3.69 Impact Factor
  • Source
    • "(Chen et al., 2011; Chen et al., 2012; Eschenbacher et al., 2012) Skeletal muscle MH/CCD (RyR1 mutation) + + + + n.d. (Brini et al., 2005; Durham et al., 2008; Giulivi et al., 2011) Amylotrophic lateral sclerosis (SOD1 mutation) + + + n.d. n.d. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mitochondria are strategically and dynamically positioned in the cell to spatially coordinate ATP production with energy needs and to allow the local exchange of material with other organelles. Interactions of mitochondria with the sarco-endoplasmic reticulum (SR/ER) have been receiving much attention owing to emerging evidence on the role these sites have in cell signaling, dynamics and biosynthetic pathways. One of the most important physiological and pathophysiological paradigms for SR/ER-mitochondria interactions is in cardiac and skeletal muscle. The contractile activity of these tissues has to be matched by mitochondrial ATP generation that is achieved, at least in part, by propagation of Ca(2+) signals from SR to mitochondria. However, the muscle has a highly ordered structure, providing only limited opportunity for mitochondrial dynamics and interorganellar interactions. This Commentary focuses on the latest advances in the structure, function and disease relevance of the communication between SR/ER and mitochondria in muscle. In particular, we discuss the recent demonstration of SR/ER-mitochondria tethers that are formed by multiple proteins, and local Ca(2+) transfer between SR/ER and mitochondria.
    Journal of Cell Science 07/2013; 126(14). DOI:10.1242/jcs.093609 · 5.43 Impact Factor
Show more