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. 2011 Oct 1;20(19):3841-51.
doi: 10.1093/hmg/ddr305. Epub 2011 Jul 12.

Muscle choline kinase beta defect causes mitochondrial dysfunction and increased mitophagy

Satomi Mitsuhashi  1 , Hideyuki Hatakeyama, Minako Karahashi, Tomoko Koumura, Ikuya Nonaka, Yukiko K Hayashi, Satoru Noguchi, Roger B Sher, Yasuhito Nakagawa, Giovanni Manfredi, Yu-ichi Goto, Gregory A Cox, Ichizo Nishino
Affiliations

Muscle choline kinase beta defect causes mitochondrial dysfunction and increased mitophagy

Satomi Mitsuhashi et al. Hum Mol Genet. .

Abstract

Choline kinase is the first step enzyme for phosphatidylcholine (PC) de novo biosynthesis. Loss of choline kinase activity in muscle causes rostrocaudal muscular dystrophy (rmd) in mouse and congenital muscular dystrophy in human, characterized by distinct mitochondrial morphological abnormalities. We performed biochemical and pathological analyses on skeletal muscle mitochondria from rmd mice. No mitochondria were found in the center of muscle fibers, while those located at the periphery of the fibers were significantly enlarged. Muscle mitochondria in rmd mice exhibited significantly decreased PC levels, impaired respiratory chain enzyme activities, decreased mitochondrial ATP synthesis, decreased coenzyme Q and increased superoxide production. Electron microscopy showed the selective autophagic elimination of mitochondria in rmd muscle. Molecular markers of mitophagy, including Parkin, PINK1, LC3, polyubiquitin and p62, were localized to mitochondria of rmd muscle. Quantitative analysis shows that the number of mitochondria in muscle fibers and mitochondrial DNA copy number were decreased. We demonstrated that the genetic defect in choline kinase in muscle results in mitochondrial dysfunction and subsequent mitochondrial loss through enhanced activation of mitophagy. These findings provide a first evidence for a pathomechanistic link between de novo PC biosynthesis and mitochondrial abnormality.

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Figures

Figure 1.
Figure 1.
Muscle histopathology. H&E staining of triceps or quadriceps femoris muscles in 8-week-old homozygous rmd mutant mice and unaffected (+/rmd or +/+) littermate controls (A–D) shows dystrophic changes including variation in fiber size, necrosis and regeneration of individual fibers and interstitial fibrosis. NADH-TR staining (E–H), immunostaining of mitochondrial outer membrane protein Tom20 and EM (M–P) show abnormal mitochondria. Mitochondria in rmd muscle fibers are enlarged and prominent at the periphery, but sparse in the center (I–L). z, Z line; m, M line; i, I band.
Figure 2.
Figure 2.
The PC level is decreased in rmd muscle mitochondria. The PE level is not altered. The PC/PE ratio is significantly decreased in rmd. The CL level is slightly decreased in rmd hindlimb. Data are expressed as the mean ± SD of eight experiments. *P < 0.01, **P < 0.0001.
Figure 3.
Figure 3.
Mitochondrial energetic function is altered and CoQ level is decreased in rmd. (A) Mitochondrial respiratory chain enzyme activities in rmd were compared with healthy littermates. C1, Complex I ; C2, Complex II ; C3, Complex III ; C4, Complex IV (n = 4). (B) The rate of ATP synthesis measured by luminometry method (n = 4). (C) Total CoQ9 level (littermate forelimb, n = 13; littermate hindlimb, n = 12; rmd forelimb, n = 11; rmd hindlimb, n = 13). Data are expressed as the mean ± SD of experiment number shown as n. *P < 0.05, **P < 0.005, ***P < 0.001.
Figure 4.
Figure 4.
Mitochondrial respiratory enzyme activity is decreased without the loss of the enzyme complex. (A) Native PAGE gel electrophoresis. In-gel activity staining shows that Complex III activity is decreased in rmd. Representative data from four different experiments are shown. (B) Immunoblotting of Complex II and III shows protein levels are maintained despite defect in significant Complex III enzymatic activity. Representative data from three different experiments of six samples are shown.
Figure 5.
Figure 5.
Mitochondrial superoxide production is increased and oxidative stress is increased in muscle tissue in rmd. (A) Mitochondrial superoxide production is enhanced in rmd, especially in hindlimb muscle mitochondria. Data are shown as the mean ± SD of seven experiments. *P < 0.001. (B) MDA levels are increased in muscle tissue. **P < 0.0005. Data are shown as the mean ± SD (n = 4 for rmd and n = 5 for littermate controls). (C) HNE4-modified proteins are increased in rmd hindlimb muscle. Coomassie brilliant blue staining is shown as a loading control. Representative data of six samples.
Figure 6.
Figure 6.
Mitochondrial degeneration in rmd. (A) EM of extensor digitorum longus muscle. In rmd, mitochondria are degraded by mitophagy. Scale bar = 0.5 μm. (B) Western blot of isolated muscle mitochondria immunodetected for Parkin, polyubiquitin, p62/SQSTM1 and LC3. TOM20, a mitochondrial outer membrane protein is used as loading control. Hindlimb mitochondria in rmd show significantly increased expression level in these mitophagy markers. (C) p62 and TOM20 immunohistrochemistry of hindlimb muscle section. Note that mitochondria are significantly enlarged and sparse in rmd. p62 colocalizes with the mitochondrial outer membrane protein TOM20. Polyubiquitin and mitochondrial protein cytochrome c oxidase (COX) colocalize. LC3 and TOM20 colocalize. Polyubiquitin and p62 colocalize. Scale bar = 10 μm.
Figure 7.
Figure 7.
(A) Mitochondrial morphometrical analysis. All mitochondria are counted in cross-sections of EDL muscle by EM. Number of mitochondria per 1 μm2 of muscle fiber cross-sectional area is shown (n = 20). The percentage of area occupied by mitochondria in a cross-section of muscle fiber is not different in rmd and littermates (n = 20). The average total mitochondrial area per muscle fiber is larger in rmd compared with littermates (n = 20). *P < 0.005, **P < 0.0005. (B) mtDNA copy number is decreased in rmd compared with littermate controls. Copy number of mtDNA (ND1) was normalized by nuclear DNA (pcam1) (M; month-old, 1 M hindlimb: rmd; n = 4, littermates; n = 4. 2 M hindlimb: rmd; n = 5, littermates; n= 6, 3 M hindlimb: rmd; n = 4, littermates; n = 6. 2 M liver: rmd; n = 5, littermates; n = 5. 2 M forelimb: rmd; n = 6, littermates; n = 6).
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