Myotubularin family phosphatase ceMTM3 is required for muscle maintenance by preventing excessive autophagy in Caenorhabditis elegans
© Yu et al.; licensee BioMed Central Ltd. 2012
Received: 14 December 2011
Accepted: 17 October 2012
Published: 31 October 2012
Autophagy is a ubiquitous cellular process responsible for the bulk degradation of cytoplasmic components through the autophagosomal-lysosomal pathway. In skeletal muscle, autophagy has been regarded as a key regulator for muscle mass maintenance, and its imbalance leads to sarcopenia. However, the underlying mechanism is poorly understood.
In this study, we demonstrate that ceMTM3, a FYVE-domain containing myotubalarin family phosphatase, is required for the maintenance of muscle fibers by preventing excessive autophagy in Caenorhabditis elegans. Knockdown of ceMTM3 by using feeding-based RNA interference caused loss of muscle fibers accompanied by shortening of muscle cell and body size in aged C. elegans worms. This was preceded by the occurrence of excessive autophagy in the muscle and other tissues, which subsequently resulted in increased lysosomal activity and necrotic cell death. However, knockdown of ceMTM3 did not aggravate the abnormalities of muscle wasting in autophagy-deficient atg-18 mutant worms.
Our data suggest an important role of ceMTM3 in regulating autophagy and maintaining muscle fibers. This study may have clinical implications for prevention and treatment of sarcopenia.
KeywordsPhosphatase Myotubalarin RNAi Autophagy Muscle C. elegans Sarcopenia
Autophagy is an evolutionarily conserved intracellular process by which cytoplasmic constituents, including long-lived proteins, protein aggregates, organelles, and invading pathogens, are delivered to lysosomes for degradation and subsequent recycling . Autophagy is activated in response to changes in the internal status of the cell and/or changes in the extracellular environment and plays an essential role in cell and tissue homeostasis. Deregulation of autophagy has been linked to many human diseases such as cancer, neurodegeneration, myopathies, diabetes, and infections by bacteria and viruses .
Genetic screens, primarily in the yeast S. cerevisiae, have identified numerous autophagy-related genes (ATG), many of which have orthologs in higher eukaryotes, including C. elegans, Drosophila, and mammals [1, 3]. The products of these genes constitute two unique protein conjugation systems responsible for formation and elongation of autophagic isolation membranes during autophagy . In addition to the conjugation systems, autophagy initiation is also dependent on phosphatidylinositol 3-phosphate (PI3P) based on the crucial role of the type III PI3Kinase Vps34, an enzyme that generates PI3P and forms a complex with autophagy regulator Beclin-1 (ATG-6) . The level of PI3P is also controlled by PI3P phosphatases that belong to the myotubularin family in mammalian cells , which presumably play a role in regulation of autophagy as well. Indeed, studies have demonstrated that PI3P phosphatase Jumpy and myotubularin-related phosphatase 3 (MTMR3) act as negative regulators of autophagy in mammalian cells [7, 8].
Myotubularin phosphatases are members of the protein tyrosine phosphatase superfamily . But unlike other protein phosphatases, myotubularin family enzymes dephosphorylate PI3P and phosphatidylinositol (3, 5)-bi-phosphate . Mutations in genes encoding myotubularin proteins are associated with diseases. For example, mutations in MTM1, the founding member of this family, cause X-linked myotubular myopathy (XLMTM), a severe congenital muscular disorder, while mutations in MTMR2 and MTMR13 are associated with Charcot-Marie-Tooth disease [9–11]. We previously have isolated and characterized a C. elegans homolog of myotubularin proteins, designated ceMTM3. ceMTM3 preferably dephosphorylates PI3P and contains a FYVE lipid-binding domain at its C-terminus which binds to PI3P . Knockdown of ceMTM3 in C. elegans worms by using feeding-based RNA interference caused severe impairment of body movement following post-reproductive age and also significantly shortened their lifespan . We reasoned that this may be related to loss of muscle function due to de-regulation of autophagy. In this study, we demonstrate that knockdown of ceMTM3 induces autophagy that precedes an accelerated loss of muscle fibers in C. elegans worms.
Results and discussion
Knockdown of ceMTM3 causes loss of muscle fibers in adult C. elegans
Knockdown of ceMTM3 shortens the body size of adult C. elegans
Knockdown of ceMTM3 causes occurrence of autophagy in multiple cells of adult C. elegans worms
Knockdown ceMTM3 increases lysosomal activities and induces necrotic cell death in C. elegans
Knockdown ceMTM3 does not aggravate the abnormalities of muscle wasting in autophagy-deficient worms
The data described above demonstrated that knockdown of a MTM family enzyme causes autophagy and subsequently causes loss of muscle fibers. As a PI3P phosphatase, ceMTM3 should play an important role in controlling the level of PI3P. The involvement of PI3P in autophagy has been demonstrated for the most part by studying the type III PI3Kinase Vps34. Vps34 is a type III PI3 kinase responsible for generation of PI3P. It forms a complex with its autophagy-regulatory partner Beclin-1 (ATG-6), thereby initiating autophagy . Vps34 also has an essential role in membrane trafficking and endocytosis, and Vps34 mutant worms display lethality and molting defects as well as alterations in the outer nuclear membrane and in the endoplasmic reticulum [20, 21]. Our current study demonstrated that knockdown of ceMTM3, a PI3P phosphatase predominately expressed in the muscle, results in autophagy which subsequently leads to loss of muscle fibers. In a sense, one may postulate that inactivation of ceMTM3 is equivalent to activation of VPS34 in regulating the autophagy process. Therefore, PI3P phosphatases and PI3 kinases play equally important roles in regulation of autophagy.
The role of autophagy in muscle wasting and maintenance has been well documented . Autophagy appears to be a double edged sword. Too much or too little of it can cause muscle weakness and atrophy. For example, excessive autophagy, induced by denervation and fasting, contributes to muscle atrophy . By demonstrating that occurrence of autophagy upon knockdown of ceMTM3 precedes muscle fiber loss, our study provides further proof of the harmful effects of too much autophagy in muscle function. On the other hand, deficiency in autophagy is also known to cause muscle atrophy. For example, both Atg5−/− and Atg7−/− mice show muscle loss accompanied by accumulation of protein aggregates and abnormal membranous structures in the muscle cells [23, 24]. By the same concept, our study demonstrated that autophagy-deficient atg-18 worms exhibited impaired body movement and loss of muscle fibers (Figure 6). In addition, an earlier study demonstrated that loss of autophagy function due to inactivation of unc-51/atg1 and bec-1/atg6/beclin1 results in small body size without affecting cell number . Our current data demonstrated that excessive autophagy associated with knockdown of ceMTM3 also causes shortening of body size with smaller muscle cells. Therefore, autophagy appears to be a dynamic process and should be kept in a balance. From a therapeutic perspective, it is important to understand whether the reduction of autophagy is helpful during muscle loss or whether enhancement of autophagosome flux is beneficial for the clearance of dangerous organelles or toxic proteins. To keep healthy muscle function, we need to keep a normal level of autophagy flux to rejuvenate organelles and to remove dysfunctional mitochondria and ER membranes but to avoid excessive autophagy which may lead to breakdown of normal muscle fibers.
The loss of muscle mass, referred to as sarcopenia, is a normal phenomenon in animals as a consequence of aging . For humans, decrease in muscle tissue begins around the age of 50 years and becomes more dramatic beyond the 60th year of life. For C. elegans worms, loss of muscle fibers and consequent impairment of body movement is clearly seen after day 15 and is much accelerated by knockdown of ceMTM3 (Figure 1). Aging is associated with a progressive decline of muscle mass, strength, and quality, but the mechanism underlying the muscle wasting remains unresolved . Studies have shown that sarcopenia is not due to lack of regenerative drive in senescent skeletal muscle , implying that the maintenance of existing muscle is crucial. Among the many factors correlating with sarcopenia during aging, oxidative stress has been extensively investigated . We believe that oxidation stress-induced inactivation of MTM family phosphatases may play a major role in the muscle wasting process. Like all the other members of the tyrosine phosphatase superfamily, MTM family enzymes are susceptible to oxidation-induced inactivation because they contain a highly reactive cysteinyl residue at the catalytic center. Reactive oxygen species, such as the superoxide radical (O2-·), hydrogen peroxide (H2O2), the hydroxyl radical (OH·), and nitric oxide (NO) are produced in muscle at rest, and this generation is increased by contractile activity . Oxidative damage is considered the main cause of aging. In fact, it was thought that a slower accumulation of oxidative damage is at least partly responsible for the life extension effects seen in C. elegans with daf-2 and age-1 mutations . Our earlier data have shown that ceMTM3 is sensitive to peroxide and loss of ceMTM3 function causes muscle deterioration in C. elegans. Therefore, inactivation of ceMTM3 by oxidation may be attributable to loss of muscle fibers in normal worms at later ages. Thus, to prevent sarcopenia, it is important to maintain a normal level of PI3P phosphatase activity.
By using the C. elegans worm as a model system, our study provides further evidence that the MTM family phosphatases play a negative role in autophagy. Our study also demonstrates a correlation between excessive autophagy and muscle wasting, implying a normal level of autophagy is important for muscle function and maintenance. Considering sensitivity of tyrosine phosphatase superfamily enzymes to reactive oxygen species, loss of muscle fibers during normal aging may be a consequence of PI3P phosphatase inactivation. Therefore, studying the MTM enzymes may have clinical implications in the prevention and treatment of human sarcopenia.
Nematode strains and maintenance
C. elegans worms were grown on nematode growth medium (NGM) plates with abundant E. coli food at 20°C according to standard protocols. Wild-type Bristol N2, ATG-18-deficient strain VC893 atg-18(gk378) V], and GFP::myosin heavy chain A-expressing RW1596 [myo-3(st386) V; stEx30] worms were obtained from the Caenorhabditis Genetics Center. Transgenic N2 worms [Plgg-1::gfp::lgg-1+rol-6] carrying the fusion protein GFP::LGG-1 autophagy marker were kindly provided by Dr. Beth Levine (University of Texas Southwestern Medical School) . All worms were scored at the same chronological age and were moved to new plates every day after they reached the reproductive period to avoid progeny contamination. Day 0 refers to the laid egg stage.
Knockdown of ceMTM3
The full-length coding sequence of ceMTM3 (−6 to 2,910 with translation starting codon ATG starting from 1) was cloned into the pPD129.36 vector. Plain p129.36 vector was used as control throughout the study. The HT115 (DE3) E. coli cells were employed as hosts for expression of double-stranded RNAs, and 0.4 mM IPTG was used to induce expression of dsRNA. The efficiency of RNAi-medicated knockdown was confirmed by western blotting with anti-ceMTM3 antibodies as previously described .
Staining of worms and microscopy
For phalloidin staining of actin filaments, worms were fixed with cold acetone and then stained with Alexa Fluor 568-phalloidin. For acridine orange staining, 500 ul of 0.01 mg/ml acridine orange made in M9 buffer was added directly onto worms cultured in a 6 cm NGM plate to ensure the entire plates were evenly covered. After incubation at 20°C in the dark for 1 h, the worms were transferred to a new culture plate without acridine orange and allowed to recover at 20°C for 1 h before being immobilized with sodium azide for microscopic analysis . For visualization of fluorescence in GFP-positive worms, worms were treated with sodium azide and then viewed under a fluorescent microscope with a GFP filter. All microscopic analysis was performed with an Olympus BX51 microscope equipped with DIC lens. Digital images were captured using an Olympus DP71 camera with the DP-BSW application software (version 03.02). Quantification of fluorescent signals was performed by using the FluorChem SP program from Alpha Innotech.
Statistical comparison between control and treatment groups was performed with unpaired t test using the GraphPad Prism software (Graphpad Software, La Jolla, CA). Differences with p<0.05 were defined significant.
Green fluorescent protein
Nematode growth medium
This work was supported by grants HL076309 and HL079441 from the National Institutes of Health (USA) and a pilot grant from the Reynolds Oklahoma Center on Aging (to ZJ Zhao) and by the Doctoral Fund of Ministry of Education of China (No. 20090061110019, to X Fu).
- Xie Z, Klionsky DJ: Autophagosome formation: core machinery and adaptations. Nat Cell Biol. 2007, 9: 1102-1109. 10.1038/ncb1007-1102.View ArticlePubMedGoogle Scholar
- Levine B, Kroemer G: Autophagy in the Pathogenesis of Disease. Cell. 2008, 132: 27-42. 10.1016/j.cell.2007.12.018.PubMed CentralView ArticlePubMedGoogle Scholar
- Kourtis N, Tavernarakis N: Autophagy and cell death in model organisms. Cell Death Differ. 2009, 16: 21-30. 10.1038/cdd.2008.120.View ArticlePubMedGoogle Scholar
- Yoshimori T, Noda T: Toward unraveling membrane biogenesis in mammalian autophagy. Curr Opin Cell Biol. 2008, 20: 401-407. 10.1016/j.ceb.2008.03.010.View ArticlePubMedGoogle Scholar
- Noda T, Matsunaga K, Taguchi-Atarashi N, Yoshimori T: Regulation of membrane biogenesis in autophagy via PI3P dynamics. Semin Cell Dev Biol. 2010, 21: 671-676. 10.1016/j.semcdb.2010.04.002.View ArticlePubMedGoogle Scholar
- Robinson FL, Dixon JE: Myotubularin phosphatases: policing 3-phosphoinositides. Trends Cell Biol. 2006, 16: 403-412. 10.1016/j.tcb.2006.06.001.View ArticlePubMedGoogle Scholar
- Vergne I, Roberts E, Elmaoued RA, Tosch V, Delgado MA, Proikas-Cezanne T, Laporte J, Deretic V: Control of autophagy initiation by phosphoinositide 3-phosphatase Jumpy. EMBO J. 2009, 28: 2244-2258. 10.1038/emboj.2009.159.PubMed CentralView ArticlePubMedGoogle Scholar
- Taguchi-Atarashi N, Hamasaki M, Matsunaga K, Omori H, Ktistakis NT, Yoshimori T, Noda T: Modulation of local PtdIns3P levels by the PI phosphatase MTMR3 regulates constitutive autophagy. Traffic. 2010, 11: 468-478. 10.1111/j.1600-0854.2010.01034.x.View ArticlePubMedGoogle Scholar
- Laporte J, Hu LJ, Kretz C, Mandel JL, Kioschis P, Coy J, Klauck SM, Poustka A, Dahl N: A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast. Nat Genet. 1996, 13: 175-182. 10.1038/ng0696-175.View ArticlePubMedGoogle Scholar
- Bolino A, Muglia M, Conforti FL, LeGuern E, Salih MA, Georgiou DM, Christodoulou K, Hausmanowa-Petrusewicz I, Mandich P, Schenone A, Gambardella A, Bono F, Quattrone A, Devoto M, Monaco AP: Charcot-Marie-Tooth type 4B is caused by mutations in the gene encoding myotubularin-related protein-2. Nat Genet. 2000, 25: 17-19. 10.1038/75542.View ArticlePubMedGoogle Scholar
- Senderek J, Bergmann C, Weber S, Ketelsen UP, Schorle H, Rudnik-Schoneborn S, Buttner R, Buchheim E, Zerres K: Mutation of the SBF2 gene, encoding a novel member of the myotubularin family, in Charcot-Marie-Tooth neuropathy type 4B2/11p15. Hum Mol Genet. 2003, 12: 349-356. 10.1093/hmg/ddg030.View ArticlePubMedGoogle Scholar
- Ma J, Zeng F, Ho WT, Teng L, Li Q, Fu X, Zhao ZJ: Characterization and functional studies of a FYVE domain-containing phosphatase in C. elegans. J Cell Biochem. 2008, 104: 1843-1852. 10.1002/jcb.21752.PubMed CentralView ArticlePubMedGoogle Scholar
- Meissner B, Warner A, Wong K, Dube N, Lorch A, McKay SJ, Khattra J, Rogalski T, Somasiri A, Chaudhry I, Fox RM, 3rd Miller DM, Baillie DL, Holt RA, Jones SJ, Marra MA, Moerman DG: An integrated strategy to study muscle development and myofilament structure in Caenorhabditis elegans. PLoS Genet. 2009, 5: e1000537-10.1371/journal.pgen.1000537.PubMed CentralView ArticlePubMedGoogle Scholar
- Sandri M: Autophagy in health and disease. 3. Involvement of autophagy in muscle atrophy. Am J Physiol Cell Physiol. 2010, 298: C1291-C1297. 10.1152/ajpcell.00531.2009.View ArticlePubMedGoogle Scholar
- Melendez A, Talloczy Z, Seaman M, Eskelinen EL, Hall DH, Levine B: Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science. 2003, 301: 1387-1391. 10.1126/science.1087782.View ArticlePubMedGoogle Scholar
- Katz DJ, Edwards TM, Reinke V, Kelly WG: A C. elegans LSD1 demethylase contributes to germline immortality by reprogramming epigenetic memory. Cell. 2009, 137: 308-320. 10.1016/j.cell.2009.02.015.PubMed CentralView ArticlePubMedGoogle Scholar
- Raben N, Shea L, Hill V, Plotz P: Monitoring autophagy in lysosomal storage disorders. Methods Enzymol. 2009, 453: 417-449.PubMed CentralView ArticlePubMedGoogle Scholar
- Samara C, Syntichaki P, Tavernarakis N: Autophagy is required for necrotic cell death in Caenorhabditis elegans. Cell Death Differ. 2008, 15: 105-112. 10.1038/sj.cdd.4402231.View ArticlePubMedGoogle Scholar
- Lu Q, Yang P, Huang X, Hu W, Guo B, Wu F, Lin L, Kovács AL, Yu L, Zhang H: The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes. Dev Cell. 2011, 21: 343-357. 10.1016/j.devcel.2011.06.024.View ArticlePubMedGoogle Scholar
- Takacs-Vellai K, Vellai T, Puoti A, Passannante M, Wicky C, Streit A, Kovacs AL, Müller F: Inactivation of the autophagy gene bec-1 triggers apoptotic cell death in C. elegans. Curr Biol. 2005, 15: 1513-1517. 10.1016/j.cub.2005.07.035.View ArticlePubMedGoogle Scholar
- Roggo L, Bernard V, Kovacs AL, Rose AM, Savoy F, Zetka M, Wymann MP, Müller F: Membrane transport in Caenorhabditis elegans: an essential role for VPS34 at the nuclear membrane. EMBO J. 2002, 21: 1673-1683. 10.1093/emboj/21.7.1673.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang X, Blagden C, Fan J, Nowak SJ, Taniuchi I, Littman DR, Burden SJ: Runx1 prevents wasting, myofibrillar disorganization, and autophagy of skeletal muscle. Genes Dev. 2005, 19: 1715-1722. 10.1101/gad.1318305.PubMed CentralView ArticlePubMedGoogle Scholar
- Raben N, Hill V, Shea L, Takikita S, Baum R, Mizushima N, Ralston E, Plotz P: Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease. Hum Mol Genet. 2008, 17: 3897-3908. 10.1093/hmg/ddn292.PubMed CentralView ArticlePubMedGoogle Scholar
- Masiero E, Agatea L, Mammucari C, Blaauw B, Loro E, Komatsu M, Metzger D, Reggiani C, Schiaffino S, Sandri M: Autophagy is required to maintain muscle mass. Cell Metab. 2009, 10: 507-515. 10.1016/j.cmet.2009.10.008.View ArticlePubMedGoogle Scholar
- Aladzsity I, Tóth ML, Sigmond T, Szabó E, Bicsák B, Barna J, Regos A, Orosz L, Kovács AL, Vellai T: Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans. Genetics. 2007, 177: 655-660. 10.1534/genetics.107.075762.PubMed CentralView ArticlePubMedGoogle Scholar
- Deschenes MR: Effects of aging on muscle fibre type and size. Sports Med. 2004, 34: 809-824. 10.2165/00007256-200434120-00002.View ArticlePubMedGoogle Scholar
- Thompson LV: Age-related muscle dysfunction. Exp Gerontol. 2009, 44: 106-111. 10.1016/j.exger.2008.05.003.PubMed CentralView ArticlePubMedGoogle Scholar
- Edström E, Ulfhake B: Sarcopenia is not due to lack of regenerative drive in senescent skeletal muscle. Aging Cell. 2005, 4: 65-77. 10.1111/j.1474-9728.2005.00145.x.View ArticlePubMedGoogle Scholar
- Rossi P, Marzani B, Giardina S, Negro M, Marzatico F: Human skeletal muscle aging and the oxidative system: cellular events. Curr Aging Sci. 2008, 1: 182-191. 10.2174/1874609810801030182.View ArticlePubMedGoogle Scholar
- Jackson MJ, McArdle A: Age-related changes in skeletal muscle reactive oxygen species generation and adaptive responses to reactive oxygen species. J Physiol. 2011, 589: 2139-2145. 10.1113/jphysiol.2011.206623.PubMed CentralView ArticlePubMedGoogle Scholar
- Honda Y, Honda S: Oxidative stress and life span determination in the nematode Caenorhabditis elegans. Ann N Y Acad Sci. 2002, 959: 466-474.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.