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Biochimica et Biophysica Acta 1801 (2010) 446–454

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a l i p

Differential expression of choline kinase isoforms in skeletal muscle explains the phenotypic variability in the rostrocaudal muscular dystrophy mouse Gengshu Wu a,b, Roger B. Sher b, Gregory A. Cox b, Dennis E. Vance a,⁎ a b

Group on the Molecular and Cell Biology of Lipids and Department of Biochemistry, University of Alberta, Edmonton, Alberta Canada T6G 2S2 The Jackson Laboratory, Bar Harbor, Maine 04609, USA

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Article history: Received 27 October 2009 Received in revised form 2 December 2009 Accepted 11 December 2009 Available online 21 December 2009 Keywords: Phosphatidylcholine Choline kinase Muscle CTP:phosphocholine cytidylyltransferase Muscular dystrophy

a b s t r a c t Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase β is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase α is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb −/− mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A2 in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb−/− mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb−/− mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb−/− mice is due to abundant choline kinase α and the stable homeostasis of phosphatidylcholine. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Muscular dystrophies are genetically heterogeneous disorders that affect 1:2000 births worldwide [1]. We recently discovered a novel muscular dystrophy with a rostral to caudal gradient resulting from disruption of the gene encoding choline kinase-ß (Chkb) [2]. Choline kinase (CK) catalyzes the first reaction in the CDP–choline pathway for phosphatidylcholine (PC) biosynthesis. Besides playing an important role in PC biosynthesis, activation of CK and the resulting increase in phosphocholine are proposed as necessary events for proliferation of fibroblasts induced by some growth factors (e.g. platelet-derived growth factor and basic fibroblast growth factor) [3]. Induction of CK is also associated with cell stress such as that caused by carcinogens in murine hepatocytes [4,5]. CK exists in mice in two isoforms, CKα and CKβ, which are encoded by the Chka and Chkb genes, respectively [4]. CKα and CKβ have distinct tissue distributions suggesting a non-redundant function for each isoform. A previous study demonstrated that CKα is crucial for the early development of mouse embryos [6]. In contrast,

Abbreviations: CK, choline kinase; CT, CTP:phosphocholine cytidylyltransferase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLC, phospholipase C; PLA2, phospholipase A2; VLDL, very low density lipoproteins ⁎ Corresponding author. Tel.: + 1 780 492 8286; fax: + 1 780 492 3383. E-mail address: [email protected] (D.E. Vance). 1388-1981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2009.12.003

genomic deletion of the Chkb gene in mice causes neonatal forelimb bone deformity and severe muscular dystrophy in hindlimbs [2]. This observation provides evidence that reduction of CK activity and tissue PC levels can result in muscular dystrophy. Since no previous evidence had linked PC biosynthesis and muscular dystrophy, this result was unanticipated. We have recently reported that the muscular dystrophy in hindlimbs of CKβ-deficient mice is due to attenuated PC biosynthesis and enhanced catabolism of PC [7]. In contrast to the severe muscular dystrophy in hindlimbs of Chkb −/− mice, the disease is not significantly developed in forelimb muscle. In the current study, therefore, we investigated why muscular dystrophy develops in hindlimbs but not forelimbs of Chkb−/ − mice. The results show that CKα contributes predominately to CK activity in forelimb muscle, while CKβ contributes mostly to CK activity in hindlimb muscle. Even though choline kinase activity in forelimb muscle of Chkb−/− mice is significantly lower than in Chkb+/+ mice, the rate of PC biosynthesis is enhanced due to increased activity of CTP:phosphocholine cytidylyltransferase (CT), the regulated and ratelimiting enzyme in the CDP–choline pathway of PC biosynthesis [8]. Concomitantly, the activity of PC-specific phospholipase C is also increased by CK deficiency in forelimb muscles. We, therefore, attribute the normal PC mass in forelimb muscle of Chkb−/− mice to an altered balance between PC biosynthesis and breakdown. Forelimb, but not hindlimb, muscles of Chkb −/− mice also had normal regenerative capacity. Furthermore, mitochondria in hindlimb muscle

G. Wu et al. / Biochimica et Biophysica Acta 1801 (2010) 446–454

from Chkb−/− mice are abnormally large and exhibit decreased inner membrane potential [2,7], whereas the presence of mega-mitochondria was not significant in forelimb muscle and mitochondrial function was normal. Therefore, the difference in occurrence of muscular dystrophy in forelimbs versus hindlimbs of CKβ deficient mice is due to the predominant contribution of CKα, but less contribution of CKβ, to total CK activity in forelimb muscle. In addition, alterations in PC biosynthesis and degradation in forelimb muscle are other important factors involved. Consequently, in forelimbs of Chkb−/− mice normal PC levels and mitochondrial function are maintained. 2. Experimental procedures 2.1. Choline kinase β-deficient mice An initial characterization of the Chkb−/− mice has been described [2]. Wild-type littermates were used as controls. Three to four-weekold mice were used in all experiments unless indicated and no gender differences were observed. Skeletal muscle from forelimbs and hindlimbs was dissected and frozen in liquid N2. All samples were stored at −70 °C before use.

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before the assay [6,7]). The amount of phosphocholine was calculated by subtracting the amount of choline from the total amount of choline metabolites quantified after alkaline phosphatase digestion. CDP– choline in the aqueous phase was identified by comparison upon thinlayer chromatography to authentic CDP–choline (Sigma, St. Louis, MO). The amount of CDP–choline was determined using a lipid phosphorus assay [14]. 2.6. Separation of water-soluble choline metabolites Total lipids were extracted from tissue homogenates as described above. The aqueous phase (water-soluble choline metabolites) was dried under nitrogen gas. The residue was re-suspended in methanol:water (1:1) and water-soluble choline metabolites were separated by thin-layer chromatography in the solvent methanol–0.5% sodium chloride–ammonia 10:10:1 (v:v). Choline, phosphocholine and CDP–choline were identified by comparison to authentic standards. Amounts of choline and phosphocholine were quantified as described above; CDP–choline was quantified using a phosphorus assay [14]. 2.7. Mitochondria from limb muscle

2.2. Lipid analysis Tissues were homogenized with a Polytron homogenizer in 5 vol of 10 mM Tris–HCl (pH 7.2), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, a protease inhibitor cocktail (1:100 dilution, Sigma, P8340). Homogenates were centrifuged for 5 min at 600 ×g and supernatants were collected. Protein was quantified by the Bradford procedure [9]. Total lipids were extracted from the supernatants [10]. Phospholipids were separated by normal phase high-performance liquid chromatography and quantified with an evaporative-light scattering detector [11]. 2.3. Enzymatic assays Muscle homogenates were centrifuged (348,000 × g for 15 min) at 4 °C. The supernatant (cytosol) and the membrane pellet were isolated. CTP:phosphocholine cytidylyltransferase (CT) activity was measured in both fractions by monitoring the conversion of [3H] phosphocholine into CDP–choline [12]. CK activity was determined in the cytosolic fraction as described [13] with minor modifications. The activity of PC-specific phospholipase C (PC-PLC) was measured using the Amplex Red PC-specific PLC assay kit from Molecular Probes (Eugene, OR). The activity of PLA2 was measured by a kit purchased from Cayman Chemical. 2.4. Immunoblot analyses Cytosolic proteins from skeletal muscle homogenates were prepared as described above. Proteins (100 µg) were separated by electrophoresis on 12% polyacrylamide gels containing 0.1% SDS and immunoblotted with rabbit antibodies to CKα (1:200; Abcam), or CKβ (1:500; a gift from Dr. K. Ishidate, Teikyo Heisei University, Japan). Tubulin (1 µg/ml, Abcam) was used as a loading control and was immunoblotted with rabbit polyclonal antibody raised against αtubulin (Abcam). 2.5. Measurement of mass of choline, phosphocholine and CDP–choline Total lipids were extracted from tissue homogenates as described above. The aqueous phase was retained and dried under N2 gas. The residue was re-suspended in water and the amount of choline determined with the Wako Phospholipids B test kit (Wako Chemicals, Japan). For measurement of the amount of [phosphocholine + choline], 2 U/ml of alkaline phosphatase was added to the sample

Muscle was homogenized in 10 mM Hepes, 0.5 mM EDTA, 0.5 mM EGTA, 250 mM sucrose and complete protease inhibitor cocktail (Roche Diagnostics) [15] using a dispersing instrument (T8.01 UltraTurrax) from IKA-WERKE (Wilmington, Delaware). The homogenate was centrifuged for 10 min at 500 ×g. The supernatant was collected and centrifuged for 10 min at 2000 ×g. The resulting pellet was washed 3× yielding a “heavy mitochondrial fraction”. The supernatant was centrifuged for 10 min at 7000×g. The pellet was washed 3× yielding the “light mitochondrial fraction”. The mitochondriaenriched pellets (of the heavy and light mitochondrial fractions) were suspended in homogenization buffer and stored at − 70 °C before use. Mitochondrial inner membrane potential was assessed using the JC-I fluorescence assay (kit from Sigma #CS0760) that measures the potential across mitochondrial inner membranes. The JC-1 dye (5, 5′, 6, 6′tetrachloro-1, 1′3, 3′-tetraethylbenzimidazolylcarbocyanine iodide). The JC-I dye concentrates in the mitochondrial matrix where it forms red fluorescent aggregates. Dissipation of the mitochondrial membrane potential prevents accumulation of JC-I dye in mitochondria. Fluorescence was detected in a fluorimeter. 2.8. Incorporation of [3H]choline into choline metabolites [methyl-3H]Choline (100 µCi) (Amersham Biosciences) in 100 µl of saline was injected into mice via the tail vein. Mice were sacrificed 1 h after injection. Blood was collected by cardiac puncture. Limb skeletal muscle was dissected, frozen in liquid N2 and stored at −70 °C before use. PC, lyso-PC, phosphocholine and CDP–choline were isolated by thin-layer chromatography [14] and radioactivity was measured. 2.9. Preparation and labeling of PC liposomes and lipoproteins Eight dishes (60 mm) of confluent McArdle RH-7777 rat hepatoma cells were incubated for 12 h with 50 µCi of [methyl-3H] choline chloride. Cells were washed 3 times and scraped into ice-cold phosphate-buffered saline. Lipids were extracted from the cells with chloroform/methanol (2:1) [10]. [3H]PC was purified by thin-layer chromatography in the solvent system chloroform/methanol/acetic acid/water (25:15:4:2, v:v) then stored at −20 °C under N2 until use. For preparation of PC liposomes, 500 µCi of [3H]PC was dried under N2 then re-suspended in 1 ml phosphate-buffered saline. The mixture was sonicated for 5 min using a tip sonicator (15-s cycles). [3H]PC-

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G. Wu et al. / Biochimica et Biophysica Acta 1801 (2010) 446–454

labeled very low density lipoproteins (VLDL) were prepared by mixing the liposomes (small unilamellar vesicles) with freshly isolated VLDL that had been isolated from healthy human donors by density gradient ultracentrifugation at 50,000 ×g for 24 h at 8 °C. The VLDL fraction (density

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