Abnormal Splicing of NEDD4 in Myotonic Dystrophy Type 2
2014; Elsevier BV; Volume: 184; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2014.04.013
ISSN1525-2191
AutoresMark Screen, Per Harald Jonson, Olayinka Raheem, Johanna Palmio, Reijo Laaksonen, Terho Lehtimäki, Mario Sirito, Ralf Krahe, Peter Hackman, Bjarne Udd,
Tópico(s)Mitochondrial Function and Pathology
ResumoMyotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG)n repeat expansion in intron 1 of CNBP. Transcription of the repeats causes a toxic RNA gain of function involving their accumulation in ribonuclear foci. This leads to sequestration of splicing factors and alters pre-mRNA splicing in a range of downstream effector genes, which is thought to contribute to the diverse DM2 clinical features. Hyperlipidemia is frequent in DM2 patients, but the treatment is problematic because of an increased risk of statin-induced adverse reactions. Hypothesizing that shared pathways lead to the increased risk, we compared the skeletal muscle expression profiles of DM2 patients and controls with patients with hyperlipidemia on statin therapy. Neural precursor cell expressed, developmentally downregulated-4 (NEDD4), an ubiquitin ligase, was one of the dysregulated genes identified in DM2 patients and patients with statin-treated hyperlipidemia. In DM2 muscle, NEDD4 mRNA was abnormally spliced, leading to aberrant NEDD4 proteins. NEDD4 was down-regulated in persons taking statins, and simvastatin treatment of C2C12 cells suppressed NEDD4 transcription. Phosphatase and tensin homologue (PTEN), an established NEDD4 target, was increased and accumulated in highly atrophic DM2 muscle fibers. PTEN ubiquitination was reduced in DM2 myofibers, suggesting that the NEDD4-PTEN pathway is dysregulated in DM2 skeletal muscle. Thus, this pathway may contribute to the increased risk of statin-adverse reactions in patients with DM2. Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG)n repeat expansion in intron 1 of CNBP. Transcription of the repeats causes a toxic RNA gain of function involving their accumulation in ribonuclear foci. This leads to sequestration of splicing factors and alters pre-mRNA splicing in a range of downstream effector genes, which is thought to contribute to the diverse DM2 clinical features. Hyperlipidemia is frequent in DM2 patients, but the treatment is problematic because of an increased risk of statin-induced adverse reactions. Hypothesizing that shared pathways lead to the increased risk, we compared the skeletal muscle expression profiles of DM2 patients and controls with patients with hyperlipidemia on statin therapy. Neural precursor cell expressed, developmentally downregulated-4 (NEDD4), an ubiquitin ligase, was one of the dysregulated genes identified in DM2 patients and patients with statin-treated hyperlipidemia. In DM2 muscle, NEDD4 mRNA was abnormally spliced, leading to aberrant NEDD4 proteins. NEDD4 was down-regulated in persons taking statins, and simvastatin treatment of C2C12 cells suppressed NEDD4 transcription. Phosphatase and tensin homologue (PTEN), an established NEDD4 target, was increased and accumulated in highly atrophic DM2 muscle fibers. PTEN ubiquitination was reduced in DM2 myofibers, suggesting that the NEDD4-PTEN pathway is dysregulated in DM2 skeletal muscle. Thus, this pathway may contribute to the increased risk of statin-adverse reactions in patients with DM2. Myotonic dystrophy type 2 (DM2; Online Mendelian Inheritance in Man 602668) is an autosomal dominant multisystemic disease with a highly variable phenotype, characterized by adult- or late-onset proximal muscle weakness, myalgia, myotonia, cardiac conduction defects, cataracts, insulin resistance, mild cerebral involvement, and liver enzyme elevation.1Machuca-Tzili L. Brook D. Hilton-Jones D. Clinical and molecular aspects of the myotonic dystrophies: a review.Muscle Nerve. 2005; 32: 1-18Crossref PubMed Scopus (197) Google Scholar, 2Krahe R. Bachinski L. Udd B. Myotonic dystrophy type 2: clinical and genetic aspects.in: Wells R.D. Ashizawa T. Genetic Instabilities and Neurological Diseases. ed 2. Academic Press/Elsevier, Amsterdam, Boston2006: 131-150Crossref Scopus (10) Google Scholar DM2 is caused by an uninterrupted (CCTG)n expansion of between 75 and 11,000 repeats in a polymorphic (TG)n(TCTG)n(CCTG)n repeat tract in intron 1 of the CNBP gene on chromosome 3q21.3Liquori C.L. Ricker K. Moseley M.L. Jacobsen J.F. Kress W. Naylor S.L. Day J.W. Ranum L.P. Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9.Science. 2001; 293: 864-867Crossref PubMed Scopus (1014) Google Scholar, 4Bachinski L.L. Czernuszewicz T. Ramagli L.S. Suominen T. Shriver M.D. Udd B. Siciliano M.J. Krahe R. Premutation allele pool in myotonic dystrophy type 2.Neurology. 2009; 72: 490-497Crossref PubMed Scopus (50) Google Scholar Typical features of DM2 muscle histopathology include extreme atrophy in a subpopulation of type IIA fibers, some of them as nuclear clump fibers, and an increased amount of internal nuclei.5Vihola A. Bassez G. Meola G. Zhang S. Haapasalo H. Paetau A. Mancinelli E. Rouche A. Hogrel J. Laforet P. Maisonobe T. Pellissier J.F. Krahe R. Eymard B. Udd B. Histopathological differences of myotonic dystrophy type 1 (DM1) and PROMM/DM2.Neurology. 2003; 60: 1854-1857Crossref PubMed Scopus (139) Google Scholar DM2 is a common form of muscular dystrophy in adults, at least in some European populations.6Suominen T. Bachinski L.L. Auvinen S. Hackman P. Baggerly K.A. Angelini C. Peltonen L. Krahe R. Udd B. Population frequency of myotonic dystrophy: higher than expected frequency of myotonic dystrophy type 2 (DM2) mutation in Finland.Eur J Hum Genet. 2011; 19: 776-782Crossref PubMed Scopus (104) Google Scholar The mutation frequency is as high as 1 in 1830 in the Finnish population,6Suominen T. Bachinski L.L. Auvinen S. Hackman P. Baggerly K.A. Angelini C. Peltonen L. Krahe R. Udd B. Population frequency of myotonic dystrophy: higher than expected frequency of myotonic dystrophy type 2 (DM2) mutation in Finland.Eur J Hum Genet. 2011; 19: 776-782Crossref PubMed Scopus (104) Google Scholar which suggests a clinical manifestation frequency of 1 in 5000. On the basis of the late onset of the symptoms, more than one-half of mutation carriers are asymptomatic at any given time. In contrast to the more severe myotonic dystrophy type 1 (DM1), there is no congenital form of DM2, and the age of onset and disease severity is not linked to the length of the repeat expansion.7Udd B. Krahe R. The myotonic dystrophies: molecular, clinical, and therapeutic challenges.Lancet Neurol. 2012; 11: 891-905Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar DM2 pathogenesis has been shown to result from an RNA gain-of-function pathomechanism that involves sequestration of trans-acting nuclear proteins, such as muscleblind-like 1, which co-localizes with mutant RNA repeats in ribonuclear foci.8Sallinen R. Vihola A. Bachinski L.L. Huoponen K. Haapasalo H. Hackman P. Zhang S. Sirito M. Kalimo H. Meola G. Horelli-Kuitunen N. Wessman M. Krahe R. Udd B. New methods for molecular diagnosis and demonstration of the (CCTG)n mutation in myotonic dystrophy type 2 (DM2).Neuromuscul Disord. 2004; 14: 274-283Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar This leads to missplicing of a range of effector genes, including INSR,9Savkur R.S. Philips A.V. Cooper T.A. Dalton J.C. Moseley M.L. Ranum L.P. Day J.W. Insulin receptor splicing alteration in myotonic dystrophy type 2.Am J Hum Genet. 2004; 74: 1309-1313Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar CLCN1,10Mankodi A. Takahashi M.P. Jiang H. Beck C.L. Bowers W.J. Moxley R.T. Cannon S.C. Thornton C.A. Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy.Mol Cell. 2002; 10: 35-44Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar BIN1,11Fugier C. Klein A.F. Hammer C. Vassilopoulos S. Ivarsson Y. Toussaint A. Tosch V. Vignaud A. Ferry A. Messaddeq N. Kokunai Y. Tsuburaya R. de la Grange P. Dembele D. Francois V. Preciquot G. Boulade-Ladame C. Hummel M.C. Lopez de Munain A. Sergeant N. Laquerriere A. Thibault C. Deryckere F. Auboeuf D. Garcia L. Zummermann P. Udd B. Schoser B. Takahashi M.P. Nishino I. Bassez G. Laporte J. Furling D. Charlet-Berquerand N. Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy.Nat Med. 2011; 17: 720-725Crossref PubMed Scopus (243) Google Scholar CACNA1S,12Tang Z.Z. Yarotskyy V. Wei L. Sobczak K. Nakamori M. Eichinger K. Moxley R.T. Dirksen R.T. Thornton C.A. Muscle weakness in myotonic dystrophy associated with misregulated splicing and altered gating of Ca(V)1.1 calcium channel.Hum Mol Genet. 2012; 21: 1312-1324Crossref PubMed Scopus (126) Google Scholar and other genes,13Vihola A. Bachinski L.L. Sirito M. Olufemi S. Hajibashi S. Baggerly K.A. Raheem O. Haapasalo H. Suominen T. Holmlund-Hampf J. Paetau A. Cardani R. Meola G. Kalimo H. Edström L. Krahe R. Udd B. Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.Acta Neuropathol. 2010; 119: 465-479Crossref PubMed Scopus (57) Google Scholar, 14Nakamori M. Sobczak K. Puwanant A. Welle S. Eichinger K. Pandya S. Dekdebrun J. Heatwole C.R. McDermott M.P. Chen T. Cline M. Tawil R. Osborne R.J. Wheeler T.M. Swanson M.S. Moxley 3rd, R.T. Thornton C.A. Splicing biomarkers of disease severity in myotonic dystrophy.Ann Neurol. 2013; 74: 862-872Crossref PubMed Scopus (172) Google Scholar which likely contribute to the phenotypic features in patients with DM2. However, other mechanisms, such as decreased CCHC-type zinc finger, nucleic acid binding protein (CNBP) expression may also have a role in the DM2 pathology.15Raheem O. Olufemi S.E. Bachinski L.L. Vihola A. Sirito M. Holmlund-Hampf J. Haapasalo H. Li Y.P. Udd B. Krahe R. Mutant (CCTG)n expansion causes abnormal expression of zinc finger protein 9 (ZNF9) in myotonic dystrophy type 2.Am J Pathol. 2010; 177: 3025-3036Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Patients who take statins have a dose-related and time-dependent increased risk of adverse muscle reactions and rhabdomyolysis.16Rallidis L.S. Fountoulaki K. Anastasiou-Nana M. Managing the underestimated risk of statin-associated myopathy.Int J Cardiol. 2011; 159: 169-176Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar There is also a clear increase in the frequency of adverse events during combination therapy or if there is an underlying subclinical myopathy.17Laaksonen R. On the mechanisms of statin-induced myopathy.Clin Pharmacol Ther. 2006; 79: 529-531Crossref PubMed Scopus (26) Google Scholar Patients with DM2 frequently have hyperlipidemia and often require statin treatment.18Udd B. Meola G. Krahe R. Thornton C. Ranum L. Bassez G. Kress W. Schoser B. Moxley R. 140th ENMC International Workshop: myotonic dystrophy DM2/PROMM and other myotonic dystrophies with guidelines on management.Neuromuscul Disord. 2006; 16: 403-413Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 19Heatwole C. Johnson N. Goldberg B. Martens W. Moxley 3rd, R. Laboratory abnormalities in patients with myotonic dystrophy type 2.Arch Neurol. 2011; 68: 1180-1184Crossref PubMed Scopus (27) Google Scholar Among the patients diagnosed with DM2 in Finland, a larger than average proportion had statin-induced adverse muscle reactions, including the occasional rhabdomyolysis. Certain polymorphisms in SLCO1B120Vladutiu G.D. Isackson P.J. SLCO1B1 variants and statin-induced myopathy.N Engl J Med. 2009; 360 (letter to the editor): 304Crossref PubMed Scopus (21) Google Scholar have been associated with an increased risk of statin-induced myopathy. Global expression profiling of skeletal muscles of patients without myopathy on statin treatment has shown changes in the calcium regulatory and the membrane repair machinery.21Draeger A. Sanchez-Freire V. Monastyrskaya K. Hoppeler H. Mueller M. Breil F. Mohaupt M.G. Babiychuk E.B. Statin therapy and the expression of genes that regulate calcium homeostasis and membrane repair in skeletal muscle.Am J Pathol. 2010; 177: 291-299Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar In addition, changes in the cholesterol metabolism pathways in muscle cell lines treated with statins have been reported.22Morikawa S. Murakami T. Yamazaki H. Izumi A. Saito Y. Hamakubo T. Kodama T. Analysis of the global RNA expression profiles of skeletal muscle cells treated with statins.J Atheroscler Thromb. 2005; 12: 121-131Crossref PubMed Scopus (58) Google Scholar Our aim was to identify molecular factors that increase the susceptibility of patients with DM2 to statin-adverse muscle reactions by comparing the expression profiles in three different muscle biopsy sets. Expression profiles of DM2 muscle biopsies were compared with previously published DM2 muscle expression profiles13Vihola A. Bachinski L.L. Sirito M. Olufemi S. Hajibashi S. Baggerly K.A. Raheem O. Haapasalo H. Suominen T. Holmlund-Hampf J. Paetau A. Cardani R. Meola G. Kalimo H. Edström L. Krahe R. Udd B. Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.Acta Neuropathol. 2010; 119: 465-479Crossref PubMed Scopus (57) Google Scholar and profiles of patients with hyperlipidemia with no muscle disorders, taking simvastatin.23Laaksonen R. Katajamaa M. Päivä H. Sysi-Aho M. Saarinen L. Junni P. Lütjohann D. Smet J. Van Coster R. Seppänen-Laakso T. Lehtimäki T. Soini J. Oresic M. A systems biology strategy reveals biological pathways and plasma biomarker candidates for potentially toxic statin-induced changes in muscle.PLoS One. 2006; 1: e97Crossref PubMed Scopus (195) Google Scholar Our hypothesis was that similar molecular pathways would be affected in patients with DM2 and by statin treatment. In this combined analysis we found 21 genes with shared dysregulated expression. In addition, the prevalence and relevance of the risk-associated polymorphism in SLCO1B1 was analyzed in a large Finnish DM2 cohort. All patients with DM1 and with DM2 were from Finland and were diagnosed by DNA mutation testing.4Bachinski L.L. Czernuszewicz T. Ramagli L.S. Suominen T. Shriver M.D. Udd B. Siciliano M.J. Krahe R. Premutation allele pool in myotonic dystrophy type 2.Neurology. 2009; 72: 490-497Crossref PubMed Scopus (50) Google Scholar Altogether nine different DM2 patient samples were studied (clinical information is summarized in Table 1). The main clinical symptoms of the patients with DM2 consisted of muscle pain and stiffness, especially after exercise, and proximal muscle weakness that was more pronounced in the lower limbs. Only two patients had clinically detectable myotonia. Half of the patients had hyperlipidemia, but none had diabetes mellitus. Whole genome expression array analysis was performed on six DM2 and four control biopsies from the vastus lateralis muscle with the use of the Illumina expression array platform (Illumina, Inc., San Diego, CA) (Table 1). For the comparison, we included previously published expression studies on DM2 patient biopsies13Vihola A. Bachinski L.L. Sirito M. Olufemi S. Hajibashi S. Baggerly K.A. Raheem O. Haapasalo H. Suominen T. Holmlund-Hampf J. Paetau A. Cardani R. Meola G. Kalimo H. Edström L. Krahe R. Udd B. Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.Acta Neuropathol. 2010; 119: 465-479Crossref PubMed Scopus (57) Google Scholar and simvastatin-treated hyperlipidemic persons.23Laaksonen R. Katajamaa M. Päivä H. Sysi-Aho M. Saarinen L. Junni P. Lütjohann D. Smet J. Van Coster R. Seppänen-Laakso T. Lehtimäki T. Soini J. Oresic M. A systems biology strategy reveals biological pathways and plasma biomarker candidates for potentially toxic statin-induced changes in muscle.PLoS One. 2006; 1: e97Crossref PubMed Scopus (195) Google Scholar Biopsies of the patients with DM2 A and B (Table 1) were used both in the Illumina DM2 array study and in the previously reported Affymetrix DM2 study.13Vihola A. Bachinski L.L. Sirito M. Olufemi S. Hajibashi S. Baggerly K.A. Raheem O. Haapasalo H. Suominen T. Holmlund-Hampf J. Paetau A. Cardani R. Meola G. Kalimo H. Edström L. Krahe R. Udd B. Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2.Acta Neuropathol. 2010; 119: 465-479Crossref PubMed Scopus (57) Google Scholar The study was approved by the institutional review board of Tampere University Hospital, and all patients gave written informed consent.Table 1Patient and Control Biopsies Used in Illumina Gene Expression Analysis, RT-PCR, and Western Blot AnalysisNameSex/age at biopsy (years)MethodMuscle symptomsClinical findingsHyperlipidemiaDiabetesMyotonic dystrophy type 2 Patient AM/49EAExercise-induced myalgiaNormal muscle strength, calf hypertrophyYesNo Patient BM/38EAExercise-induced myalgia and weaknessClinical myotonia, mild proximal lower limb atrophyYesNo Patient C (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.1)F/34EAMuscle stiffness, clumsinessNormal muscle strength, myotonia on EMGNoNo Patient D (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.2)F/41EADifficulties in climbing stairs, muscle stiffnessProximal lower limb weakness, clinical myotoniaNoNo Patient E (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.3)M/55EAExercise-induced myalgia, muscle stiffnessProximal weakness and atrophy, calf hypertrophy, myotonia on EMGNoNo Patient F (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.4)M/37EAMuscle stiffnessMild proximal weakness, myotonia on EMGYesInsulin resistance DM2-A (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.5)M/44PCRExercise-induced myalgia and weakness, muscle stiffnessMild proximal weaknessNANo DM2-B (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.4)M/37PCRSee patient F DM2-CF/63PCRMild bent spine syndromeAxial and proximal weaknessYesNo DM2-DM/51PCRDifficulties in climbing stairs, muscle stiffnessProximal weakness, myotonia on EMGNANo D1 (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.3)M/55WBSee patient E D2 (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.1)F/34WBSee patient C D3 (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.2)F/41WBSee patient D D4 (∗Overlapping biopsies indicating the same patient's biopsy was present in different experiments.5)M/44WBSee DM2-AControls Ctrl AM/52EA Ctrl BM/45EA Ctrl CM/50EA Ctrl DM/54EA C-1M/79PCR C-2M/80PCR C-3F/UPCR C1U/>65WB C2U/>65WBMyotonic dystrophy type 1 DM1-AM/34PCR DM1-BF/42PCR DM1-CM/50PCR DM1-DF/47PCRF, female; M, male; Ctrl, control; DM1, myotonic dystrophy type 1, DM2, myotonic dystrophy type 2; EA, expression array; EMG, electromyography; NA, not available; U, unknown; WB, Western blot analysis.∗ Overlapping biopsies indicating the same patient's biopsy was present in different experiments. Open table in a new tab F, female; M, male; Ctrl, control; DM1, myotonic dystrophy type 1, DM2, myotonic dystrophy type 2; EA, expression array; EMG, electromyography; NA, not available; U, unknown; WB, Western blot analysis. Muscle biopsies were homogenized with ultra-turrax (IKA turrax, S8N-5 G), and total RNA was extracted with Trizol (15596-018; Invitrogen, Carlsbad, CA) and purified with the RNeasy kit (74106; Qiagen, Valencia, CA). The total RNA was treated with DNase (79254; Qiagen) according to the manufacturer's recommendations. One hundred nanograms of total RNA was amplified and biotinylated with the use of the Illumina RNA Total Prep Amplification kit (AMIL1791; Ambion, Austin, TX) for 14 hours. The Illumina gene expression array analysis was performed at the Microarray Center at Turku University by using 1.50 μg of sample RNA with Sentrix Human-6 Expression BeadChips version 2 (BD-25-113; Illumina, Inc.) at 58°C overnight (17 hours) according to Illumina whole genome gene expression with IntelliHyb Seal-protocol, revision B. The hybridization was detected with 1 μg/mL cyanine 3–streptavidine (PA43001; GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The expression arrays were scanned with Illumina BeadArray Reader and analyzed with Bead Studio version 3. The expression array raw intensity signals were analyzed with Inforsense KDE version 2.0.4 (Inforsense, London, UK) by using quantile normalization. This software was used for single-gene analyses, including fold-change calculations and filtering of the probes. Statistical significance was calculated with an unpaired t-test. Probes that did not reach statistical significance (P ≤ 0.05) were removed from the data. The microarray data are available from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo; accession number GSE45331). Pathway changes were identified with the Database for Annotation, Visualization and Integrated Discovery ontology database (http://david.abcc.ncifcrf.gov, last accessed January 24, 2011) with KEGG (Kyoto Encyclopedia of Genes and Genomes) downstream annotations. Significant pathway enrichment in the DM2 expression data were calculated with EASE score (modified Fisher exact t-test) with multiple testing correction (Benjamini–Hochberg). The pathways were ranked with the EASE score t-test (P ≤ 0.05). Genomic DNA was extracted from peripheral blood leukocytes by using the QIAamp DNA Blood Mini-kit. The SLCO1B1 single nucleotide polymorphism (SNP) rs4149056 was genotyped with TaqMan genotyping assays (Applied Biosystems, Foster City, CA). Random duplicates were used as controls. cDNA was generated with 1 μg of total RNA from myotube cultures or from muscle tissue by using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. In vitro transcription was performed with random hexamers and oligo-(dT) priming according to the manufacturer's instructions (SuperScript III First-strand cDNA Synthesis Kit; Invitrogen). The PCR products were amplified with DreamTaq master mix (EP0702; Fermentas, Burlington, ON, Canada) and were separated on an agarose gel. PCR products were identified by sequencing of representative DNA bands. Primer sequences are given in Table 2.Table 2A List of Primers Used in RT-PCR AnalysisOligonucleotideSequenceNEDD4 006-1L5′-CTCCTCCTCCTCCACAGTTG-3′NEDD4 006-1R5′-CGGTGCTGCTGAGGATGA-3′NEDD4Fwd: 5′-TTGCAGCAACAACAAGAACC-3′Rev: 5′-GCAAGTCCAGGCGATTAAAA-3′Nedd4Fwd: 5′-CACAGTGGAAAAGGCCAAGC-3′Rev: 5′-ACCTGGTGGCAATCCAGATG-3′GAPDHFwd: 5′-TTAGCACCCCTGGCCAAGG-3′Rev: 5′-CTTACTCCTTGGAGGCCATG-3′Fwd, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NEDD4, neural precursor cell expressed, developmentally down-regulated 4, E3 ubiquitin protein ligase; Rev, reverse. Open table in a new tab Fwd, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NEDD4, neural precursor cell expressed, developmentally down-regulated 4, E3 ubiquitin protein ligase; Rev, reverse. Quantification of the cDNA was performed with TaqMan-based quantitative real-time PCR by using NEDD4 (Hs00406454_m1), Gapdh (Mm99999915_g1), and GAPDH (4333764F) primers and probes (Life Technologies, Carlsbad, CA). TaqMan master mix (10 mL; Applied Biosystems), 0.5 μL of 1:10 diluted cDNA, and 2 μL of primer and probe sets were used in a 20-μL total reaction volume. Amplification and detection were performed with the ABI 7500 system (Applied Biosystems). The PCR thermal conditions were 50°C for 2 minutes, 95°C for 10 seconds, and 60°C for 1 minute. Each sample was performed in triplicate and normalized to GAPDH by using standard curves for each gene on the same plate. Muscle biopsies were prepared for SDS-PAGE by homogenization with 19 volumes of sample buffer that contained 4 mol/L urea and 4% SDS at 100°C for 5 minutes. Samples were resolved with 12% SDS-PAGE gels with 4% stacking gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. Antibodies used in immunoblotting, immunofluorescence (IF), and immunohistochemistry (IHC) are rabbit monoclonal anti–phosphatase and tensin homologue (PTEN; IF and IHC dilution 1:100; 6H2.1; Millipore, Billerica, MA), rabbit polyclonal anti-ubiquitin (dilution 1:150; Z0458; Dako UK Ltd., Ely, UK), mouse anti-dystrophin (dilution 1:50; Dy4/6D3; Novocastra, Newcastle, UK), polyclonal rabbit anti-NEDD4 (dilution 1:1000; NBP1-03462; Novus Biologicals, Inc., Littleton, CO), mouse anti-GAPDH (dilution 1:20,000; ab8245; Abcam, Cambridge, MA), and rabbit monoclonal anti-MBNL1 (dilution 1:1000; ab108519; Abcam). Primary antibodies were detected with secondary horseradish peroxidase–conjugated antibodies (dilution 1:100; DAKO P260; DakoCytomation, Glostrup, Denmark) and enhanced chemiluminescence with the Immun-Star kit (Bio-Rad Laboratories). Secondary horseradish peroxidase–conjugated antibodies were diluted at 1:1000 in Western blot analysis. Cell culture of biopsies was performed as described previously.15Raheem O. Olufemi S.E. Bachinski L.L. Vihola A. Sirito M. Holmlund-Hampf J. Haapasalo H. Li Y.P. Udd B. Krahe R. Mutant (CCTG)n expansion causes abnormal expression of zinc finger protein 9 (ZNF9) in myotonic dystrophy type 2.Am J Pathol. 2010; 177: 3025-3036Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Immunoprecipitation (IP) assays of PTEN were done according to the manufacturer's instructions (Pierce cross-linking IP kit; Thermo Fisher Scientific, Inc., Waltham, MA). PTEN was captured with the anti-PTEN antibody (dilution 1:100; ab32199; Abcam) and identified by Western blot analysis as described above. DM2 muscle biopsies (n = 6) and control muscle biopsies (n = 2) were snap frozen in liquid nitrogen-cooled isopentane for 6 μm cryosections on SuperFrost. IHC staining was performed with the NovoLink Min Polymer detection system (reference RE7290-K; Leica Microsystems, Milton Keynes, UK) by using the aforementioned antibodies. IF was performed as previously described15Raheem O. Olufemi S.E. Bachinski L.L. Vihola A. Sirito M. Holmlund-Hampf J. Haapasalo H. Li Y.P. Udd B. Krahe R. Mutant (CCTG)n expansion causes abnormal expression of zinc finger protein 9 (ZNF9) in myotonic dystrophy type 2.Am J Pathol. 2010; 177: 3025-3036Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar by using Alexa Fluor 680 donkey anti-mouse (A10038; Invitrogen) and Alexa Fluor 405 goat anti-rabbit (A31556; Invitrogen) secondary antibodies (dilution 1:1000). Fluorescence was detected with an Axioplan Imager 2 microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) with a high-resolution, cooled camera. AxioVision version 4.6 (Carl Zeiss) was used for image acquisition. Simvastatin (S6196; Sigma-Aldrich, St. Louis, MO) was converted into the active acid according to the manufacturer's instructions. C2C12 myoblasts were grown in Dulbecco's modified Eagle's medium (Gibco 41965; Invitrogen) with high glucose (4.5 g/L) and supplemented with 10% fetal bovine serum, 50 U/mL penicillin, and 50 μg/mL streptomycin. Myoblasts were seeded in T25 flasks for 2 days until fully confluent and differentiated in Dulbecco's modified Eagle's medium with high glucose that was supplemented with penicillin, streptomycin, and 2% horse serum. After 1 week simvastatin-containing media were added to the myotubes (no treatment, ethanol vehicle, 1 μmol/L, and 10 μmol/L) in quadruplicate. The cells were harvested after 6 hours, and RNA was extracted with an RNeasy kit (Qiagen). cDNA was then produced, and PCR products were amplified with mouse primers. Cells were also harvested after 2 days of simvastatin treatment, and total protein was extracted for Western blot analysis. Band intensities were quantified with ImageJ version 1.46f (NIH, Bethesda, MD). Significance of changes between treatment groups was calculated with a two-tailed equal variance t-test. Plasmids for shRNA expression that target all protein-coding isoforms of MBNL1 and a scramble control were purchased from GeneCopoeia (Rockville, MD). Two combined shRNAs were used that contained the target sequence n7 (5′-CAATTGCAACCGAGGAGAA-3′) or n8 (5′-AGATCAAGGCTGCCCAATA-3′) in the HSH011081-7-mU6 vector. HEK293T cells were plated on 6-well plates at 150,000 cells per well and transfected with 2 μg of combined MBNL1 shRNA plasmids or a scramble control (CSHC TR001-mU6) by using FuGene 6 (Promega, Madison, WI) and OptiMem (Life Technologies) according to the manufacturer's instructions. Cells were harvested 72 hours after transfection for Western blot analysis and RNA extraction. Eight patients referred to the Neuromuscular Research Center (Tampere University and University Hospital, Finland) because of simvastatin-induced muscle symptoms (muscle pain/weakness and elevated levels of creatine kinase, including one case of rhabdomyolysis) proved to have DM2 on genetic examination. In the same 2004 to 2007 period, a total of 89 patients were diagnosed with DM2. Twelve of these patients were diagnosed in family studies, 77 were primary referrals of undetermined muscle disease, and among them were the eight patients with simvastatin-adverse reactions. Thus, 10% of the primary referred patients who proved to have DM2 had simvastatin-induced symptoms as the main cause of referral. During the 2008 to 2009 period, 19 further patients were referred for neuromuscular evaluation because of statin-induced muscle symptoms, of which 4 patients (21%) had a subsequent genetic diagnosis of DM2. Genotyping of the known myopathy-associated SNP rs4149056 in SLCO1B120Vladutiu G.D. Isackson P.J. SLCO1B1 variants and statin-induced myopathy.N Engl J Med. 2009; 360 (
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