Title: Basal Bioenergetic Abnormalities in Skeletal Muscle from Ryanodine Receptor Malignant Hyperthermia-susceptible R163C Knock-in Mice
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 Ca2+ 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 Ca2+, 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 Ca2+-responsive signaling pathways regulated by calcineurin and ERK1/2. Chronically elevated resting Ca2+ 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. 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 Ca2+ 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 Ca2+, 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 Ca2+-responsive signaling pathways regulated by calcineurin and ERK1/2. Chronically elevated resting Ca2+ 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. IntroductionMalignant hyperthermia (MH) 2The abbreviations used are: MHmalignant hyperthermiaROSreactive oxygen speciesMHSMH-susceptibleCCDcentral core diseaseCncalcineurinqPCRquantitative PCRPMpost-mitochondrialCYTBcytochrome bNFATnuclear factor of activated T-cellSSMsubsarcolemmalRCRrespiratory control ratioICP-MSinductively coupled plasma mass spectrometryAMPKAMP-dependent protein kinaseACCacetyl-CoA carboxylaseSRsarcoplasmic reticulum. is an inherited pharmacogenetic disorder of skeletal muscle characterized by an abnormal response to muscle-depolarizing relaxants such as succinylcholine and volatile anesthetics (1Denborough M.A. Forster J.F. Lovell R.R. Maplestone P.A. Villiers J.D. Br. J. 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Genet. 2009; 10: 104Crossref PubMed Scopus (85) Google Scholar).Genotype-phenotype correlations associated with mutations in the RYR1 gene are complex and may be partly explained by how mutations in different regions of the RyR1 protein influence conformation and functional regulation of the channel. Subtle functional differences among the large number of mutations currently known could also explain why a subset of mutations confer MH susceptibility without clinical evidence of early onset CCD, whereas others confer MH and CCD of varying severity (15Davis M.R. Haan E. Jungbluth H. Sewry C. North K. Muntoni F. Kuntzer T. Lamont P. Bankier A. Tomlinson P. Sánchez A. Walsh P. Nagarajan L. Oley C. Colley A. Gedeon A. Quinlivan R. Dixon J. James D. Müller C.R. Laing N.G. Neuromuscul. Disord. 2003; 13: 151-157Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 20Monnier N. Romero N.B. Lerale J. Landrieu P. Nivoche Y. Fardeau M. Lunardi J. Hum. Mol. 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How these aspects of Ca2+ dysregulation influence respiratory parameters and mitochondrial functions in the nontriggered (basal) state is poorly understood.More than 40 years ago, it was suggested that uncoupling of oxidative phosphorylation could explain the metabolic disturbances seen in MH (33Wilson R.D. Traber D.L. Allen C.R. Priano L.L. South. Med. J. 1971; 64: 411-414Crossref PubMed Google Scholar). However, other laboratories were not able to show that halothane uncoupling of oxidative phosphorylation could explain the rapid rise in body temperature seen in MH (34Wang J.K. Moffitt E.A. Rosevear J.W. Anesthesiology. 1969; 30: 439-442Crossref PubMed Google Scholar). Furthermore, no difference was detected in isolated mitochondria from control and MH patients during halothane exposures (35Britt B.A. Kalow W. Gordon A. Humphrey J.G. Rewcastle N.B. Can. Anaesth. Soc. J. 1973; 20: 431-467Crossref PubMed Google Scholar, 36Britt, B. 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We investigated the effect of this RyR1 mutation on mitochondria obtained from skeletal muscle, in which the expression of full-length RyR1 is found, and its downstream Ca2+-dependent effectors are tailored to suit the distinctive function of this organ.DISCUSSIONContraction of skeletal muscle depends on the increase of intracellular Ca2+ concentrations, which are initiated by the action potential. Myoplasmic Ca2+ can vary from 0.12 μm under resting conditions to as much as 1 μm during contraction or 10 μm in contractures (109Marban E. Rink T.J. Tsien R.W. Tsien R.Y. Nature. 1980; 286: 845-850Crossref PubMed Scopus (148) Google Scholar). Considering that the R163C RyR1 mutation in skeletal muscle resulted in a significantly increased cytosolic Ca2+ (about 2-fold of the normal resting concentration, this study and that by Yang et al. (30Yang T. Esteve E. Pessah I.N. Molinski T.F. Allen P.D. López J.R. Am. J. Physiol. 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Our results indicate that skeletal muscle mitochondria from R163C-RyR1 mice have accumulated more calcium within their matrix (presumably mainly as Ca2+-phosphate precipitate (110Argaud L. Gateau-Roesch O. Augeul L. Couture-Lepetit E. Loufouat J. Gomez L. Robert D. Ovize M. Am. J. Physiol. Heart Circ. Physiol. 2008; 294: H386-H391Crossref PubMed Scopus (0) Google Scholar)) than controls under basal conditions (i.e. in the absence of a fulminant MH episode). One possible caveat is that EGTA used in the isolation buffer could have reduced the free Ca2+ within mitochondria thereby resulting in an underestimate of mitochondrial calcium. Despite this possible limitation, we were able to identify significant differences in total matrix calcium between genotypes. Considering that a subpopulation of mitochondria has been found closely attached to SR (87Rizzuto R. Brini M. Murgia M. Pozzan T. Science. 1993; 262: 744-747Crossref PubMed Google Scholar, 88Rizzuto R. Pinton P. Carrington W. 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Aracena-Parks P. Long C. Rossi A.E. Goonasekera S.A. Boncompagni S. Galvan D.L. Gilman C.P. Baker M.R. Shirokova N. Protasi F. Dirksen R. Hamilton S.L. Cell. 2008; 133: 53-65Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar), and mitochondria loss, known to occur in CCD skeletal muscle fibers (2Dubowitz V. Pearse A.G. Lancet. 1960; 2: 23-24Abstract PubMed Google Scholar).However, chronically elevated resting Ca2+ in R163C skeletal muscle elicited the maintenance of a fast-twitch fiber program with clear mitochondrial defects. These defects included decreased mitochondrial number and altered mitochondrial function in permeabilized muscle and intact muscle, with the development of an insulin resistance-like phenotype as part of a metabolic adaptation to the R163C RyR1 mutation.Several possibilities may explain the differences in state 3-dependent oxygen uptake between WT and R163C mice. First, the mitochondrial pellet may be contaminated with nonmitochondrial protein (i.e. myosin, fragmented myofibrils, or myoglobin). Any contaminating protein is assayed in the BCA reaction (56Wiechelman K.J. Braun R.D. Fitzpatrick J.D. Anal. Biochem. 1988; 175: 231-237Crossref PubMed Scopus (597) Google Scholar), resulting in an underestimation of the oxygen consumption rate when related to mitochondrial protein concentration. The normalization of oxygen uptake rates to a mitochondrial enzyme such as cytochrome c oxidase rather than to mitochondrial protein may eliminate this problem in normal studies, but it is not appropriate in diseased muscle where specific mitochondrial enzymes may be selectively depressed. The presence of a significant contamination of nonmitochondrial protein was excluded in our study based on the low recovery of both a highly abundant protein such as myoglobin (0.03 and 0.08% in WT and R163C; Fig. 1C) and the oligomycin-resistant ATPase activity (111Fernandez J.L. Rosemblatt M. Hidalgo C. Biochim. Biophys. Acta. 1980; 599: 552-568Crossref PubMed Google Scholar, 112Harigaya S. Ogawa Y. Sugita H. J. Biochem. 1968; 63: 324-331PubMed Google Scholar) (1.6 and 2.0% in WT and R163C) in the mitochondrial fractions. These results supported the notion that the contamination from cytosolic (myoglobin) or SR/plasmalemma (oligomycin-resistant ATPase) was negligible (<1% on average).A second possibility is that slow respiratory rates with all substrates with loose coupling, as in the case with R163C (TABLE 1, TABLE 2), may indicate a damaged preparation. However, the biochemical characteristics of muscle mitochondria from WT, run in parallel to those of R163C, were of similar or higher quality than those published by others (Table 1) suggesting that this is not the case.Another possible explanation is that during mitochondrial isolation, homogenization of diseased muscle may free a higher percentage of total muscle mitochondrial fractions with a higher proportion of damaged organelles, resulting in a falsely low respiration rate. If this hypothesis is correct, oxygen uptake rates should be lowest in preparations with higher mitochondrial protein yields. This alternative can also be excluded because the mitochondrial protein yield from R163C and WT mice was not significantly different (Table 1), whereas the state 3-dependent oxygen uptake in R163C was 62 ± 3% of WT (Table 2). In addition, data on the maximal oxygen uptake sustained by NADH obtained with permeabilized muscle to test for mitochondrial function in situ was 52 ± 9% that of WT, similar to the differences seen with isolated mitochondria supplemented with an NAD-linked substrate (Table 2). The slight discrepancy between these numbers could be explained by considering that all mitochondrial populations are assayed in permeabilized muscle with limited cytoskeletal disruption, whereas the preparation with isolated mitochondria is likely to be more enriched with intact, less dysfunctional organelles (113Andrienko T. Kuznetsov A.V. Kaambre T. Usson Y. Orosco A. Appaix F. Tiivel T. Sikk P. Vendelin M. Margreiter R. Saks V.A. J. Exp. 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Tandler B. Loffredo F. Vazquez E. Hoppel C. Am. J. Physiol. Heart Circ. Physiol. 2005; 289: H868-H872Crossref PubMed Scopus (0) Google Scholar, 118Ritov V.B. Menshikova E.V. Azuma K. Wood R. Toledo F.G. Goodpaster B.H. Ruderman N.B. Kelley D.E. Am. J. Physiol. Endocrinol. Metab. 2010; 298: E49-E58Crossref PubMed Scopus (0) Google Scholar), then the presence of dysfunctional SSM in R163C could be result in an insulin resistance-like phenotype.Taken together, these data support the hypothesis that the R163C RyR1 mutation in skeletal muscle results in defects in mitochondrial number and function. Moreover, the mutation also elicited lower glucose utilization by both OXPHOS and glycolysis (Table 3). To gain insight into the interaction between pathways, we used the DAVID gene functional classification tool (67Dennis Jr., G. Sherman B.T. Hosack D.A. Yang J. Gao W. Lane H.C. Lempicki R.A. Genome Biol. 2003; 4: P3Crossref PubMed Google Scholar, 68Huang da W. Sherman B.T. Lempicki R.A. Nat. Protoc. 2009; 4: 44-57Crossref PubMed Scopus (20924) Google Scholar) to condense the list of pathways enriched in the proteins and/or transcripts found altered in R163C mice. The resulting distribution highlighted overrepresented Gene Ontology categories. Besides the OXPHOS pathway, others that ranked high in the hierarchy were the following: Cn/NFAT, insulin, and calcium and muscle contraction (Fig. 5).The specific regulation of slow fiber-specific genes is controlled by the Cn pathway (95Chin E.R. Olson E.N. Richardson J.A. Yang Q. Humphries C. Shelton J.M. Wu H. Zhu W. Bassel-Duby R. Williams R.S. Genes Dev. 1998; 12: 2499-2509Crossref PubMed Google Scholar). Cn, a Ca2+-activated, calmodulin-dependent protein Ser/Thr phosphatase that senses intracellular Ca2+ levels, dephosphorylates NFAT, which translocates to the nucleus and regulates the transcription of target genes (95Chin E.R. Olson E.N. Richardson J.A. Yang Q. Humphries C. Shelton J.M. Wu H. Zhu W. Bassel-Duby R. Williams R.S. Genes Dev. 1998; 12: 2499-2509Crossref PubMed Google Scholar). Transgenic mice overexpressing an activated form of Cn in skeletal muscle presented a marked shift in glucose (decreased glucose oxidation with increased glycogen formation) and lipid (increased fatty acid oxidation and mitochondria biogenesis) metabolism via coordinated expression of metabolic genes, as well as transcription regulators, including peroxisome proliferator-activated receptor δ, peroxisome proliferator-activated receptor α, and PGC1-α (96Long Y.C. Glund S. Garcia-Roves P.M. Zierath J.R. J. Biol. Chem. 2007; 282: 1607-1614Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), with increased expression of slow contractile machinery (97Naya F.J. Mercer B. Shelton J. Richardson J.A. Williams R.S. Olson E.N. J. Biol. Chem. 2000; 275: 4545-4548Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Conversely, pharmacological inhibition of Cn activity induces a slow to fast myosin ATPase transformation in rat soleus muscle (95Chin E.R. Olson E.N. Richardson J.A. Yang Q.