Impaired Intracellular Energetic Communication in Muscles from Creatine Kinase and Adenylate Kinase (M-CK/AK1) Double Knock-out Mice
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m303150200
ISSN1083-351X
AutoresEdwin Janssen, André Terzic, Bé Wieringa, Petras P. Dzeja,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoPreviously we demonstrated that efficient coupling between cellular sites of ATP production and ATP utilization, required for optimal muscle performance, is mainly mediated by the combined activities of creatine kinase (CK)- and adenylate kinase (AK)-catalyzed phosphotransfer reactions. Herein, we show that simultaneous disruption of the genes for the cytosolic M-CK- and AK1 isoenzymes compromises intracellular energetic communication and severely reduces the cellular capability to maintain total ATP turnover under muscle functional load. M-CK/AK1 (MAK=/=) mutant skeletal muscle displayed aberrant ATP/ADP, ADP/AMP and ATP/GTP ratios, reduced intracellular phosphotransfer communication, and increased ATP supply capacity as assessed by 18O labeling of [Pi] and [ATP]. An analysis of actomyosin complexes in vitro demonstrated that one of the consequences of M-CK and AK1 deficiency is hampered phosphoryl delivery to the actomyosin ATPase, resulting in a loss of contractile performance. These results suggest that MAK=/= muscles are energetically less efficient than wild-type muscles, but an apparent compensatory redistribution of high-energy phosphoryl flux through glycolytic and guanylate phosphotransfer pathways limited the overall energetic deficit. Thus, this study suggests a coordinated network of complementary enzymatic pathways that serve in the maintenance of energetic homeostasis and physiological efficiency. Previously we demonstrated that efficient coupling between cellular sites of ATP production and ATP utilization, required for optimal muscle performance, is mainly mediated by the combined activities of creatine kinase (CK)- and adenylate kinase (AK)-catalyzed phosphotransfer reactions. Herein, we show that simultaneous disruption of the genes for the cytosolic M-CK- and AK1 isoenzymes compromises intracellular energetic communication and severely reduces the cellular capability to maintain total ATP turnover under muscle functional load. M-CK/AK1 (MAK=/=) mutant skeletal muscle displayed aberrant ATP/ADP, ADP/AMP and ATP/GTP ratios, reduced intracellular phosphotransfer communication, and increased ATP supply capacity as assessed by 18O labeling of [Pi] and [ATP]. An analysis of actomyosin complexes in vitro demonstrated that one of the consequences of M-CK and AK1 deficiency is hampered phosphoryl delivery to the actomyosin ATPase, resulting in a loss of contractile performance. These results suggest that MAK=/= muscles are energetically less efficient than wild-type muscles, but an apparent compensatory redistribution of high-energy phosphoryl flux through glycolytic and guanylate phosphotransfer pathways limited the overall energetic deficit. Thus, this study suggests a coordinated network of complementary enzymatic pathways that serve in the maintenance of energetic homeostasis and physiological efficiency. In tissues with high and sudden energy demand, creatine kinase (CK) 1The abbreviations used are: CK, creatine kinase; AK, adenylate kinase; ScCKmit, skeletal muscle containing an additional mitochondrial CK isoform; NDPK, nucleoside diphosphokinase; GPS, gastrocnemius-plantaris-soleus; CrP, creatine phosphate; Glc-6-P, glucose 6-phosphate; PEP, phosphoenolpyruvate; MM-CK, muscle-type dimeric cytosolic CK.1The abbreviations used are: CK, creatine kinase; AK, adenylate kinase; ScCKmit, skeletal muscle containing an additional mitochondrial CK isoform; NDPK, nucleoside diphosphokinase; GPS, gastrocnemius-plantaris-soleus; CrP, creatine phosphate; Glc-6-P, glucose 6-phosphate; PEP, phosphoenolpyruvate; MM-CK, muscle-type dimeric cytosolic CK.- and adenylate kinase (AK)-catalyzed reactions form the principal pathways securing efficient communication between the subcellular compartments responsible for production and utilization of metabolic energy (1Wallimann T. Wyss M. Brdiczka D. 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Creatine kinases (CK, EC 2.7.3.2) catalyzing the reaction MgADP– + CrP2– + H+ ↔ Cr + MgATP2– belong to a smaller and evolutionary younger family of enzymes with a role in high energy phosphoryl transfer and cellular energy buffering (1Wallimann T. Wyss M. Brdiczka D. Nicolay K. Eppenberger H.M. Biochem. J. 1992; 281: 21-40Crossref PubMed Scopus (1573) Google Scholar, 5Bessman S.P. Carpenter C.L. Annu. Rev. Biochem. 1985; 54: 831-862Crossref PubMed Scopus (580) Google Scholar, 24van Deursen J. Heerschap A. Oerlemans F. Ruitenbeek W. Jap P. ter Laak H. Wieringa B. Cell. 1993; 74: 621-631Abstract Full Text PDF PubMed Scopus (276) Google Scholar). Creatine kinases are foremost found in cells with high peak demands in metabolic energy such as the brain, heart, or skeletal muscle (1Wallimann T. Wyss M. Brdiczka D. Nicolay K. Eppenberger H.M. Biochem. J. 1992; 281: 21-40Crossref PubMed Scopus (1573) Google Scholar, 5Bessman S.P. Carpenter C.L. Annu. Rev. Biochem. 1985; 54: 831-862Crossref PubMed Scopus (580) Google Scholar). In skeletal muscle, the principal CK isoform is the cytosolic isoform, MM-CK, a homodimer mainly present as a soluble protein in the cytosol and bound to the myofibrillar M- and I-bands (13Wegmann G. Zanolla E. Eppenberger H.M. Wallimann T. J. Muscle Res. Cell Motil. 1992; 13: 420-435Crossref PubMed Scopus (80) Google Scholar) as well as to the sarcoplasmic reticulum membranes (25Rossi A.M. Eppenberger H.M. Volpe P. Cotrufo R. Wallimann T. J. Biol. Chem. 1990; 265: 5258-5266Abstract Full Text PDF PubMed Google Scholar). Skeletal muscle also contains an additional mitochondrial CK isoform (ScCKmit), which amounts to 1–10% of the total CK activity depending on the type of muscle fiber (26Wyss M. Smeitink J. Wevers R.A. Wallimann T. Biochim. Biophys. Acta. 1992; 1102: 119-166Crossref PubMed Scopus (374) Google Scholar, 27Veksler V.I. Kuznetsov A.V. Anflous K. Mateo P. van Deursen J. Wieringa B. Ventura Clapier R. J. 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Physiol. 1998; 84: 1166-1173Crossref PubMed Scopus (19) Google Scholar). By pharmacological inhibition of the CK circuit, it has been demonstrated that an increase in AK-mediated phosphotransfer may compensate for the loss of CK-activity (17Dzeja P.P. Zeleznikar R.J. Goldberg N.D. J. Biol. Chem. 1996; 271: 12847-12851Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Likewise, in skeletal muscles carrying a null mutation in either the M-CK or AK1 gene, leading to complete lack of corresponding protein expression and activity, an adaptive rewiring of flux through the remaining intact phosphotransfer circuit occurs (6Janssen E. Dzeja P.P. Oerlemans F. Simonetti A.W. Heerschap A. de Haan A. Rush P.S. Terjung R.R. Wieringa B. Terzic A. EMBO J. 2000; 19: 6371-6381Crossref PubMed Scopus (127) Google Scholar, 18Dzeja P.P. Zeleznikar R.J. Goldberg N.D. Mol. Cell. Biochem. 1998; 184: 169-182Crossref PubMed Google Scholar, 19Pucar D. Janssen E. Dzeja P.P. Juranic N. Macura S. 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Wijers M. Fransen J. Dzeja P.P. Terzic A. Wieringa B. J. Biol. Chem. 2003; 278: 12937-12945Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Although progress has been made in our understanding of individual phosphotransfer reactions, the consequences of combined deletion of major AK and CK isoforms remain unknown. Here, we report on the effects deleting both the AK1 and M-CK proteins in a single cell-type skeletal muscle fiber of a double knock-out mouse. Use of this model provides us with a unique opportunity to assess the significance of the activities of mitochondrial CK, glycolytic enzymes, and NDPK-mediated phosphotransfer reactions in muscle physiology and metabolism. Monitoring intracellular phosphotransfer kinetics by [18O]phosphoryl labeling revealed that lack of AK1 and M-CK resulted in a serious impairment of communication between ATP-generating and ATP-consuming cellular sites. As a consequence, AK1/M-CK-deficient muscles had a reduced ability to sustain the dynamic fluctuations in ATP/ADP/AMP nucleotide metabolism and overall cellular ATP turnover during functional load despite increased high energy phosphoryl flux through alternative glycolytic and guanylate phosphotransfer pathways. These data provide further evidence for the existence of a fully integrated high energy phosphoryl transfer system with a high degree of functional plasticity required for optimal muscle energetics. Generation of M-CK/AK1-deficient Mice—Gene-targeted AK1-deficient mice were derived from mouse embryonic stem cells carrying a replacement mutation in the AK1 gene. Creatine kinase knock-out mice were derived from embryonic stem cells carrying a replacement mutation in the M-CK gene. Cohorts of AK1 –/– and M-CK –/– animals were generated and maintained as previously described (24van Deursen J. Heerschap A. Oerlemans F. Ruitenbeek W. Jap P. ter Laak H. Wieringa B. Cell. 1993; 74: 621-631Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 39Janssen E. De Groof A. Wijers M. Fransen J. Dzeja P.P. Terzic A. Wieringa B. J. Biol. Chem. 2003; 278: 12937-12945Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). AK1 and M-CK knock-out mice were cross-bred to obtain animals that were heterozygous for both AK1 and M-CK alleles, and these animals were subsequently mated to obtain homozygous double mutants. Genotyping for wild-type and mutant AK1 and M-CK alleles was performed using a PCR assay as described previously (6Janssen E. Dzeja P.P. Oerlemans F. Simonetti A.W. Heerschap A. de Haan A. Rush P.S. Terjung R.R. Wieringa B. Terzic A. EMBO J. 2000; 19: 6371-6381Crossref PubMed Scopus (127) Google Scholar, 40Steeghs K. Oerlemans F. de Haan A. Heerschap A. Verdoodt L. de Bie M. Ruitenbeek W. Benders A. Jost C. van Deursen J. Tullson P. Terjung R. Jap P. Jacob W. Pette D. Wieringa B. Mol. Cell. Biochem. 1998; 184: 183-194Crossref PubMed Google Scholar). Lack of AK1 and M-CK protein activity was confirmed by Western blot and zymogram analysis. Gastrocnemius and soleus muscles from age matched homozygous M-CK/AK1-deficient (MAK=/=) and wild-type control animals also having a 50–50% C57BL/6 × 129/Ola-mixed inbred background were used for all of the studies. The investigation conformed to the Guidelines for the Care and Use of Laboratory Animals of the Dutch Council and the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee at the University of Nijmegen and the Mayo Clinic. High energy Phosphoryl Transfer—ATP turnover and phosphoryl flux through AK, CK, and glycolytic systems were measured in intact gastrocnemius-plantaris-soleus (GPS) muscle complex using the [18O]phosphoryl labeling technique (16Zeleznikar R.J. Dzeja P.P. Goldberg N.D. J. Biol. Chem. 1995; 270: 7311-7319Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 17Dzeja P.P. Zeleznikar R.J. Goldberg N.D. J. Biol. Chem. 1996; 271: 12847-12851Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21Dzeja P.P. Vitkevicius K.T. Redfield M.M. Burnett J.C. Terzic A. Circ. Res. 1999; 84: 1137-1143Crossref PubMed Scopus (175) Google Scholar, 41Zeleznikar R.J. Goldberg N.D. J. Biol. Chem. 1991; 266: 15110-15119Abstract Full Text PDF PubMed Google Scholar). Mice were anesthetized with pentobarbital (Beuthanasia D) (100 mg/kg intraperitoneal) and injected with heparin (50 units of intraperitoneal) prior to muscle dissection. Isolated mouse GPS muscle was preincubated for 12 min at room temperature in a medium containing (in mm) 137 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 1 NaH2PO4, 20 HEPES, 0.05 EDTA, 5 glucose, and 24 NaHCO3 (pH 7.4). The muscle complex was then rapidly transferred into a medium enriched with 30% 18O-water (Isotec Inc.) and paced at 2 Hz exactly as described previously (6Janssen E. Dzeja P.P. Oerlemans F. Simonetti A.W. Heerschap A. de Haan A. Rush P.S. Terjung R.R. Wieringa B. Terzic A. EMBO J. 2000; 19: 6371-6381Crossref PubMed Scopus (127) Google Scholar). After 3 min of 18O labeling, muscles were freeze-clamped using liquid N2 and extracted in a solution containing 0.6 m HClO4 and 1 mm EDTA. Protein content was determined using a protein assay kit (Bio-Rad). Cellular ATP, ADP, GTP, GDP, inorganic phosphate, creatine phosphate (CrP), and glucose 6-phosphate (Glc-6-P) were purified and quantified using high performance liquid chromatography (17Dzeja P.P. Zeleznikar R.J. Goldberg N.D. J. Biol. Chem. 1996; 271: 12847-12851Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 41Zeleznikar R.J. Goldberg N.D. J. Biol. Chem. 1991; 266: 15110-15119Abstract Full Text PDF PubMed Google Scholar). To obtain information on basal levels and turnover rates of phosphoryl-containing metabolites, isolated mouse GPS muscle was treated identically but without pacing. Samples containing phosphoryls of γ-ATP, β-ATP, β-ADP, γ-GTP, β-GTP, β-GDP, inorganic phosphate, and CrP as glycerol 3-phosphate were converted to trimethylsilyl derivatives. 18O enrichment of phosphoryls in glycerol 3-phosphates was determined with a Hewlett-Packard 5980B gas chromatograph mass spectrometer (6Janssen E. Dzeja P.P. Oerlemans F. Simonetti A.W. Heerschap A. de Haan A. Rush P.S. Terjung R.R. Wieringa B. Terzic A. EMBO J. 2000; 19: 6371-6381Crossref PubMed Scopus (127) Google Scholar). High Energy Phosphoryl Transfer Rates—Total cellular ATP turnover was estimated from the total number of 18O atoms that appeared in phosphoryls of Pi, CrP, γ-ATP, β-ATP, β-ADP, γ-GTP, β-GTP/GDP, and Glc-6-P. Net AK-, CK-, and hexokinase-catalyzed phosphotransfers were determined from the rate of appearance of 18O-containing phosphoryls in β-ATP and ADP and CrP and Glc-6-P, respectively (6Janssen E. Dzeja P.P. Oerlemans F. Simonetti A.W. Heerschap A. de Haan A. Rush P.S. Terjung R.R. Wieringa B. Terzic A. EMBO J. 2000; 19: 6371-6381Crossref PubMed Scopus (127) Google Scholar, 17Dzeja P.P. Zeleznikar R.J. Goldberg N.D. J. Biol. Chem. 1996; 271: 12847-12851Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 41Zeleznikar R.J. Goldberg N.D. J. Biol. Chem. 1991; 266: 15110-15119Abstract Full Text PDF PubMed Google Scholar, 42Zeleznikar R.J. Heyman R.A. Graeff R.M. Walseth T.F. Dawis S.M. Butz E.A. Goldberg N.D. J. Biol. Chem. 1990; 265: 300-311Abstract Full Text PDF PubMed Google Scholar). Metabolite Levels—ATP, ADP, GTP, and GDP levels were quantified in muscle perchloric extracts by use of high pressure liquid chromatography (21Dzeja P.P. Vitkevicius K.T. Redfield M.M. Burnett J.C. Terzic A. Circ. Res. 1999; 84: 1137-1143Crossref PubMed Scopus (175) Google Scholar, 41Zeleznikar R.J. Goldberg N.D. J. Biol. Chem. 1991; 266: 15110-15119Abstract Full Text PDF PubMed Google Scholar). AMP, ADP, CrP, muscle lactate, and glucose 6-phosphate levels were determined using coupled enzyme assays (21Dzeja P.P. Vitkevicius K.T. Redfield M.M. Burnett J.C. Terzic A. Circ. Res. 1999; 84: 1137-1143Crossref PubMed Scopus (175) Google Scholar, 24van Deursen J. Heerschap A. Oerlemans F. Ruitenbeek W. Jap P. ter Laak H. Wieringa B. Cell. 1993; 74: 621-631Abstract Full Text PDF PubMed Scopus (276) Google Scholar). Muscle inorganic phosphate level was determined using the EnzChek phosphate assay kit (Molecular Probes). Zymogram Analysis and Enzyme Activity—Homogenates from freshly excised GPS muscles (10% w/v) were prepared in SETH buffer (in mm: 250 sucrose, 2 EDTA, 10 Tris-HCl (pH 7.4)) at 4 °C. GPS extracts were diluted 1:1 in 30 mm Na3PO4 buffer (pH 7.4) containing 0.05% v/v Triton X-100, 0.3 mm dithiothreitol, and a complete protease inhibitor mixture (Roche Applied Science). Muscle extracts were incubated for 30 min at room temperature and centrifuged for 20 min at 11,000 × g, and an aliquot (1–5 μl) was applied to agarose gels (Alameda, CA). AK1 and creatine kinase isoenzymes were separated electrophoretically and stained for enzyme activity (6Janssen E. Dzeja P.P. Oerlemans F. Simonetti A.W. Heerschap A. de Haan A. Rush P.S. Terjung R.R. Wieringa B. Terzic A. EMBO J. 2000; 19: 6371-6381Crossref PubMed Scopus (127) Google Scholar, 43Steeghs K. Benders A. Oerlemans F. de Haan A. Heerschap A. Ruitenbeek W. Jost C. van Deursen J. Perryman B. Pette D. Bruckwilder M. Koudijs J. Jap P. Veerkamp J. Wieringa B. Cell. 1997; 89: 93-103Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). CK and AK activities were measured from whole hind limb muscle extracts (6Janssen E. Dzeja P.P. Oerlemans F. Simonetti A.W. Heerschap A. de Haan A. Rush P.S. Terjung R.R. Wieringa B. Terzic A. EMBO J. 2000; 19: 6371-6381Crossref PubMed Scopus (127) Google Scholar) using a CK NAC (N-acetyl-l-cysteine)-activated kit (Roche Applied Science) and a coupled enzyme system, respectively (21Dzeja P.P. Vitkevicius K.T. Redfield M.M. Burnett J.C. Terzic A. Circ. Res. 1999; 84: 1137-1143Crossref PubMed Scopus (175) Google Scholar). Western Blot Analysis—Skeletal muscles were excised, pulverized with a mortar and pestle using liquid N2, and extracted in a buffer containing 50 mm NaCl, 60 mm Tris-HCl (pH 7.5), 5 mm EDTA, and 0.2% Triton X-100 to which a complete protease inhibitor mixture was added. Extracts were centrifuged (10 min, 8000 × g, 4 °C), and proteins were separated on 10% SDS-polyacrylamide gels before being electrophoretically transferred onto nitrocellulose membranes. M-CK and AK1 proteins were detected using antibodies raised against chicken M-CK (44Wallimann T. Moser H. Eppenberger H.M. J. Muscle Res. Cell Motil. 1983; 4: 429-441Crossref PubMed Scopus (41) Google Scholar) and mouse AK1-glutathione S-transferase fusion proteins (15Janssen, E., Kuiper, J., Hodgson, D., Zingman, L. V., Alekseev, A. E., Terzic, A., and Wieringa, B. (2003) Mol. Cell. Biochem., in pressGoogle Scholar). Aldolase antibody (Rockland, Gilbertsville, PA) was used as a control. Immunocomplexes were visualized by chemiluminescence using a secondary antibody coupled to horseradish peroxidase and exposure to Kodak X-Omat AR films. Actomyosin Contraction—Actomyosin contraction was measured using an established superprecipitation method (45Honig C.R. Am. J. Physiol. 1968; 214: 357-364Crossref PubMed Scopus (13) Google Scholar, 46Reddy Y.S. Wyborny L. Lewis R.M. Schwartz A. Cardiovasc. Res. 1976; 10: 129-135Crossref PubMed Scopus (15) Google Scholar). Excised femoral quadriceps muscles (muscles from two mice combined) were pulverized with mortar and pestle using liquid N2 and extracted in a buffer containing 0.6 m KCl, 0.04 m NaHCO3, 0.01 m Na2CO3, 4 mm dithiothreitol, and a complete protease inhibitor mixture. Extracts were homogenized (3 × 5 s) with a blender (Polytron), maintained at 4 °C for 10–15 min, and centrifuged (30 min, 9500 × g, 4 °C). The supernatant was gently diluted with 10 volumes of ice-cold water containing 0.5 mm dithiothreitol and maintained at 4 °C for 30 min. The solution was centrifuged (15 min, 7000 × g,4 °C), and the precipitate dissolved in a buffer containing 0.6 m KCl, 20 mm Tris-HCl (pH 7.4), and the complete protease inhibitor mixture. Skeletal muscle actomyosin was stored at –20 °C after mixing with an equal volume of glycerol. Superprecipitation was measured in a buffer containing 0.8 mg/ml skeletal muscle actomyosin, 0.8 mm CaCl2, 2 mm MgCl2, 50 mm KCl, 1 mm EGTA, and 20 mm Tris-HCl (pH 6.8) at 25 °C. The change in absorbance at 660 nm was followed after the addition of ADP (0.2 or 2 mm), phosphoenolpyruvate (PEP; 2 mm), CrP (2.0 mm), or ATP (2 mm). Three measurements for each genotype (muscles from 2 mice/sample combined) were performed and statistically compared. Statistics—Data are presented as mean ± S.E. Student's t test for unpaired samples was used for statistical analysis, and p < 0.05 was considered significant. Generation of M-CK/AK1 Double Gene Knock-out Mice—To generate a mouse model lacking cytosolic AK1 and M-CK isoforms, single mutant animals carrying a homozygous null mutation in the AK1 gene (6Janssen E. Dzeja P.P. Oerlemans F. Simonetti A.W. Heerschap A. de Haan A. Rush P.S. Terjung R.R. Wieringa B. Terzic A. EMBO J. 2000; 19: 6371-6381Crossref PubMed Scopus (127) Google Scholar) or the M-CK gene (24van Deursen J. Heerschap A. Oerlemans F. Ruitenbeek W. Jap P. ter Laak H. Wieringa B. Cell. 1993; 74: 621-631Abstract Full Text PDF PubMed Scopus (276) Google Scholar) were mated. Subsequent cross-breeding of the F1 males and females with the heterozygous M-CK +/–/AK1 +/– genotype gave litter with normal size. Among an offspring of 175 pups analyzed, 9 animals were M-CK–/– and AK1–/– (hereon referred to as MAK=/=), indicating normal Mendelian segregation (1 in 16 expected). Zymogram and Western blot analysis confirmed complete absence of M-CK and AK1 protein and enzymatic activity in these animals (Fig. 1). MAK=/= mice showed no overt abnormalities, bred normally when maintained as a separate lineage over multiple (now >6) generations, and had normal life expectancy. Cellular Energetics in Resting and Contracting MAK =/= Muscle—Genetic deletion of M-CK and AK1 produced a dramatic reduction in the amount of 18O labeling of the pools of CrP, β-ATP, and β-ADP in skeletal muscle. Compared with wild type, 18O-metabolic labeling of CrP was decreased from 8.8 ± 0.4 to 5.4 ± 0.2% in MAK=/= resting non-contracting muscle, a reduction of 39% (p < 0.05, n = 6) (Fig. 2A). Stimulation of contractile activity further aggravated the difference in metabolic labeling of CrP between wild-type and mutant muscles, changing from 17.2 ± 1.2% in wild-type to 8.0 ± 0.5% in knock-out muscle (p < 0.05, n = 6), a reduction of 54% (Fig. 2A). On average, muscle stimulation at 2 Hz increased 18O labeling of CrP by 95% in wild type but gave only a 49% elevation in MAK=/= mutant muscle. This increase in [18O]CrP labeling despite the absence of M-CK could be attribu
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