Different Mouse Models of Nemaline Myopathy Harboring Acta1 Mutations Display Differing Abnormalities Related to Mitochondrial Biology
2023; Elsevier BV; Volume: 193; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2023.06.008
ISSN1525-2191
AutoresJennifer Tinklenberg, Rebecca A. Slick, J. R. Sutton, Liwen Zhang, Hui Meng, Margaret Beatka, Mark A. Vanden Avond, Mariah J. Prom, Emily Ott, Federica Montanaro, James S. Heisner, R. Toro, Edna C. Hardeman, Aron M. Geurts, David F. Stowe, R. Blake Hill, Michael W. Lawlor,
Tópico(s)Muscle Physiology and Disorders
ResumoACTA1 encodes skeletal muscle-specific α-actin, which polymerizes to form the thin filament of the sarcomere. Mutations in ACTA1 are responsible for approximately 30% of nemaline myopathy (NM) cases. Previous studies of weakness in NM have focused on muscle structure and contractility, but genetic issues alone do not explain the phenotypic heterogeneity observed in patients with NM or NM mouse models. To identify additional biological processes related to NM phenotypic severity, proteomic analysis was performed using muscle protein isolates from wild-type mice in comparison to moderately affected knock-in (KI) Acta1H40Y and the minimally affected transgenic (Tg) ACTA1D286G NM mice. This analysis revealed abnormalities in mitochondrial function and stress-related pathways in both mouse models, supporting an in-depth assessment of mitochondrial biology. Interestingly, evaluating each model in comparison to its wild-type counterpart identified different degrees of mitochondrial abnormality that correlated well with the phenotypic severity of the mouse model. Muscle histology, mitochondrial respiration, electron transport chain function, and mitochondrial transmembrane potential were all normal or minimally affected in the TgACTA1D286G mouse model. In contrast, the more severely affected KI.Acta1H40Y mice displayed significant abnormalities in relation to muscle histology, mitochondrial respirometry, ATP, ADP, and phosphate content, and mitochondrial transmembrane potential. These findings suggest that abnormal energy metabolism is related to symptomatic severity in NM and may constitute a contributor to phenotypic variability and a novel treatment target. ACTA1 encodes skeletal muscle-specific α-actin, which polymerizes to form the thin filament of the sarcomere. Mutations in ACTA1 are responsible for approximately 30% of nemaline myopathy (NM) cases. Previous studies of weakness in NM have focused on muscle structure and contractility, but genetic issues alone do not explain the phenotypic heterogeneity observed in patients with NM or NM mouse models. To identify additional biological processes related to NM phenotypic severity, proteomic analysis was performed using muscle protein isolates from wild-type mice in comparison to moderately affected knock-in (KI) Acta1H40Y and the minimally affected transgenic (Tg) ACTA1D286G NM mice. This analysis revealed abnormalities in mitochondrial function and stress-related pathways in both mouse models, supporting an in-depth assessment of mitochondrial biology. Interestingly, evaluating each model in comparison to its wild-type counterpart identified different degrees of mitochondrial abnormality that correlated well with the phenotypic severity of the mouse model. Muscle histology, mitochondrial respiration, electron transport chain function, and mitochondrial transmembrane potential were all normal or minimally affected in the TgACTA1D286G mouse model. In contrast, the more severely affected KI.Acta1H40Y mice displayed significant abnormalities in relation to muscle histology, mitochondrial respirometry, ATP, ADP, and phosphate content, and mitochondrial transmembrane potential. These findings suggest that abnormal energy metabolism is related to symptomatic severity in NM and may constitute a contributor to phenotypic variability and a novel treatment target. Nemaline myopathy (NM) affects approximately 1 in 50,0001North K.N. Laing N.G. Wallgren-Pettersson C. 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Genotype-phenotype correlations in ACTA1 mutations that cause congenital myopathies.Neuromuscul Disord. 2009; 19: 6-16Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar or the degree of nemaline rod pathology.7Malfatti E. Romero N.B. Nemaline myopathies: state of the art.Rev Neurol (Paris). 2016; 172: 614-619Crossref PubMed Scopus (46) Google Scholar Mutations in the ACTA1 gene represent a major cause of NM, as they are responsible for approximately 30% of NM cases.5Agrawal P.B. Strickland C.D. Midgett C. Morales A. Newburger D.E. Poulos M.A. Tomczak K.K. Ryan M.M. Iannaccone S.T. Crawford T.O. Laing N.G. Beggs A.H. Heterogeneity of nemaline myopathy cases with skeletal muscle alpha-actin gene mutations.Ann Neurol. 2004; 56: 86-96Crossref PubMed Scopus (118) Google Scholar ACTA1 encodes skeletal muscle-specific α-actin (ACT1A) monomers, which polymerize to form F-actin,8Sparrow J.C. Nowak K.J. Durling H.J. Beggs A.H. Wallgren-Pettersson C. Romero N. Nonaka I. 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Myopathy mutations in alpha-skeletal-muscle actin cause a range of molecular defects.J Cell Sci. 2004; 117: 3367-3377Crossref PubMed Scopus (75) Google Scholar and impaired sarcomeric function10Joureau B. de Winter J.M. Conijn S. Bogaards S.J.P. Kovacevic I. Kalganov A. Persson M. Lindqvist J. Stienen G.J.M. Irving T.C. Ma W. Yuen M. Clarke N.F. Rassier D.E. Malfatti E. Romero N.B. Beggs A.H. Ottenheijm C.A.C. Dysfunctional sarcomere contractility contributes to muscle weakness in ACTA1-related nemaline myopathy (NEM3).Ann Neurol. 2018; 83: 269-282Crossref PubMed Scopus (22) Google Scholar have all been implicated in muscle weakness due to ACTA1 mutations but do not fully explain the range of muscle weakness observed in NM. Mouse models with dominant missense mutations in Acta1 have been modeled after human mutations, including the knock-in (KI) Acta1H40Y mouse11Nguyen M.A. Joya J.E. Kee A.J. Domazetovska A. Yang N. Hook J.W. Lemckert F.A. Kettle E. Valova V.A. Robinson P.J. North K.N. Gunning P.W. Mitchell C.A. Hardeman E.C. Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy.Brain. 2011; 134: 3516-3529Crossref PubMed Scopus (50) Google Scholar and the transgenic (Tg) ACTA1D286G mouse.12Ravenscroft G. Jackaman C. Bringans S. Papadimitriou J.M. Griffiths L.M. McNamara E. Bakker A.J. Davies K.E. Laing N.G. Nowak K.J. Mouse models of dominant ACTA1 disease recapitulate human disease and provide insight into therapies.Brain. 2011; 134: 1101-1115Crossref PubMed Scopus (51) Google Scholar Both mutations cause severe NM in humans, as the patient with the ACTA1 (D286G) variant died at 9 months of age and the patient with the ACTA1 (H40Y) variant died at 2 months of age.13Nowak K.J. Wattanasirichaigoon D. Goebel H.H. Wilce M. Pelin K. Donner K. Jacob R.L. Hubner C. Oexle K. Anderson J.R. Verity C.M. North K.N. Iannaccone S.T. Muller C.R. Nurnberg P. Muntoni F. Sewry C. Hughes I. Sutphen R. Lacson A.G. Swoboda K.J. Vigneron J. Wallgren-Pettersson C. Beggs A.H. Laing N.G. Mutations in the skeletal muscle alpha-actin gene in patients with actin myopathy and nemaline myopathy.Nat Genet. 1999; 23: 208-212Crossref PubMed Scopus (349) Google Scholar Mouse models with these mutations display more subtle phenotypes. The KI.Acta1H40Y mutation affects the actomyosin interface in approximately 40% of the actin protein,14Chan C. Fan J. Messer A.E. Marston S.B. Iwamoto H. Ochala J. Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function.Biochim Biophys Acta. 2016; 1862: 1453-1458Crossref PubMed Scopus (12) Google Scholar which alters the molecular force production and produces a moderately severe model of NM with significant weakness and spontaneous death in a subset of mice.11Nguyen M.A. Joya J.E. Kee A.J. Domazetovska A. Yang N. Hook J.W. Lemckert F.A. Kettle E. Valova V.A. Robinson P.J. North K.N. Gunning P.W. Mitchell C.A. Hardeman E.C. Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy.Brain. 2011; 134: 3516-3529Crossref PubMed Scopus (50) Google Scholar,14Chan C. Fan J. Messer A.E. Marston S.B. Iwamoto H. Ochala J. Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function.Biochim Biophys Acta. 2016; 1862: 1453-1458Crossref PubMed Scopus (12) Google Scholar,15Tinklenberg J. Meng H. Yang L. Liu F. Hoffmann R.G. Dasgupta M. Allen K.P. Beggs A.H. Hardeman E.C. Pearsall R.S. Fitts R.H. Lawlor M.W. Treatment with ActRIIB-mFc produces myofiber growth and improves lifespan in the Acta1 H40Y murine model of nemaline myopathy.Am J Pathol. 2016; 186: 1568-1581Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar In the TgACTA1D286G mouse model, approximately 20% of the protein is mutated by affecting the actin-actin interface,12Ravenscroft G. Jackaman C. Bringans S. Papadimitriou J.M. Griffiths L.M. McNamara E. Bakker A.J. Davies K.E. Laing N.G. Nowak K.J. Mouse models of dominant ACTA1 disease recapitulate human disease and provide insight into therapies.Brain. 2011; 134: 1101-1115Crossref PubMed Scopus (51) Google Scholar,16Fan J. Chan C. McNamara E.L. Nowak K.J. Iwamoto H. Ochala J. Molecular consequences of the myopathy-related d286g mutation on actin function.Front Physiol. 2018; 9: 1756Crossref PubMed Scopus (6) Google Scholar which produces a subtler effect on myosin binding than the KI.Acta1H40Y model. The TgACTA1D286G mice still exhibit mild NM tissue pathology but display minimal weakness on behavioral testing and have a normal lifespan.12Ravenscroft G. Jackaman C. Bringans S. Papadimitriou J.M. Griffiths L.M. McNamara E. Bakker A.J. Davies K.E. Laing N.G. Nowak K.J. Mouse models of dominant ACTA1 disease recapitulate human disease and provide insight into therapies.Brain. 2011; 134: 1101-1115Crossref PubMed Scopus (51) Google Scholar,16Fan J. Chan C. McNamara E.L. Nowak K.J. Iwamoto H. Ochala J. Molecular consequences of the myopathy-related d286g mutation on actin function.Front Physiol. 2018; 9: 1756Crossref PubMed Scopus (6) Google Scholar,17Tinklenberg J.A. Siebers E.M. Beatka M.J. Meng H. Yang L. Zhang Z. Ross J.A. Ochala J. Morris C. Owens J.M. Laing N.G. Nowak K.J. Lawlor M.W. Myostatin inhibition using mRK35 produces skeletal muscle growth and tubular aggregate formation in wild type and TgACTA1D286G nemaline myopathy mice.Hum Mol Genet. 2018; 27: 638-648Crossref PubMed Scopus (23) Google Scholar To identify cellular processes that were abnormal across the heterogeneous genetic causes of NM, proteomic analysis of several NM mouse models was performed to identify secondarily affected pathways for further study. In comparing proteomic data sets from the KI.Acta1H40Y, TgACTA1D286G, and nebulin conditional knockout (Neb cKO) mice, abnormal protein content related to metabolism was identified to some extent in all models. A comprehensive analysis of the metabolism of Neb cKO mice is described elsewhere, and significant findings included severe deficiencies of mitochondrial respiration and ATP content, along with marked abnormalities of mitochondrial distribution and the content and localization of key metabolic regulatory proteins.18Slick R.A. Tinklenberg J.A. Sutton J. Zhang L. Meng H. Beatka M.J. Vanden Avond M. Prom M.J. Ott E. Montanaro F. Heisner J. Toro R. Granzier H. Geurts A.M. Stowe D.F. Hill R.B. Lawlor M.W. Aberrations in energetic metabolism and stress-related pathways contribute to pathophysiology in the Neb conditional knockout mouse model of nemaline myopathy.Am J Pathol. 2023; 193: 1528-1547Abstract Full Text Full Text PDF Scopus (1) Google Scholar This report describes the findings of a comprehensive proteomic and metabolic assessment of the TgACTA1D286G and KI.Acta1H40Y mouse models to provide a comparison of metabolic phenotypes in NM. The data support the notion of metabolic dysfunction in NM mice that display significant muscle weakness, which suggests a role for metabolism in contributing to the spectrum of symptomatic severity observed in NM. All studies using animal tissue were approved by the Institutional Animal Care and Use Committee at the Medical College of Wisconsin (Milwaukee, WI). Genotyping was performed as previously described.19Tinklenberg J.A. Siebers E.M. Beatka M.J. Fickau B.A. Ayres S. Meng H. Yang L. Simpson P. Granzier H.L. Lawlor M.W. Myostatin inhibition using ActRIIB-mFc does not produce weight gain or strength in the nebulin conditional KO mouse.J Neuropathol Exp Neurol. 2019; 78: 130-139Crossref PubMed Scopus (9) Google Scholar For all KI.Acta1H40Y studies, wild-type (WT; WT.Acta1H40Y) and KI (KI.Acta1H40Y) littermates were used for comparisons. For all TgACTA1D286G studies, C57Bl/6J (C57) mice were used as WT control animals because the TgACTA1D286G was generated by site-directed mutagenesis on CBA × C57Bl/6 mouse embryos to produce the human ACTA1 protein with the D286G mutation.12Ravenscroft G. Jackaman C. Bringans S. Papadimitriou J.M. Griffiths L.M. McNamara E. Bakker A.J. Davies K.E. Laing N.G. Nowak K.J. Mouse models of dominant ACTA1 disease recapitulate human disease and provide insight into therapies.Brain. 2011; 134: 1101-1115Crossref PubMed Scopus (51) Google Scholar Assessments were performed at early and late disease stages, as defined below, with the goal of identifying whether specific findings were associated with disease progression. As the goal of proteomics studies was a comparison across models, each model was assessed at the common time points of 6 and 16 weeks of age. As the goal of the histologic and functional testing was the establishment of progression-related phenotypes within a model, different time points were identified for TgACTA1D286G and KI.Acta1H40Y models to reflect their differences in severity and progression. An early disease stage time point corresponded to a time where weakness was present but not severe. For the TgACTA1D286G mice, 9 weeks of life was selected; and 6 weeks of life was selected for the KI.Acta1H40Y as a subpopulation of male KI.Acta1H40Y mice die at approximately 6 weeks of life because of complications of bladder outlet obstruction. The late disease stage time point (16 weeks of life for TgACTA1D286G mice and 9 weeks of life for KI.Acta1H40Y mice) corresponded to a time point several weeks later when weakness and pathologic severity were known to have worsened.15Tinklenberg J. Meng H. Yang L. Liu F. Hoffmann R.G. Dasgupta M. Allen K.P. Beggs A.H. Hardeman E.C. Pearsall R.S. Fitts R.H. Lawlor M.W. Treatment with ActRIIB-mFc produces myofiber growth and improves lifespan in the Acta1 H40Y murine model of nemaline myopathy.Am J Pathol. 2016; 186: 1568-1581Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar,17Tinklenberg J.A. Siebers E.M. Beatka M.J. Meng H. Yang L. Zhang Z. Ross J.A. Ochala J. Morris C. Owens J.M. Laing N.G. Nowak K.J. Lawlor M.W. Myostatin inhibition using mRK35 produces skeletal muscle growth and tubular aggregate formation in wild type and TgACTA1D286G nemaline myopathy mice.Hum Mol Genet. 2018; 27: 638-648Crossref PubMed Scopus (23) Google Scholar Both male and female mice were used in experiments but because significant differences between sexes were not observed, animals were pooled for statistical analysis. Animals were euthanized using CO2 and cervical dislocation. Triceps, gastrocnemius, and quadriceps muscles were removed, weighed, and frozen in liquid nitrogen–cooled isopentane.20Meng H. Janssen P.M. Grange R.W. Yang L. Beggs A.H. Swanson L.C. Cossette S.A. Frase A. Childers M.K. Granzier H. Gussoni E. Lawlor M.W. Tissue triage and freezing for models of skeletal muscle disease.J Vis Exp. 2014; : 51586PubMed Google Scholar Liquid nitrogen was used to grind frozen quadriceps muscle tissue (n = 5 per genotype), and 1 mL of 10% trichloroacetic acid was added per 100 mg tissue in acetone containing 2% mercaptoethanol. This was mixed by inverting the tube 10 times; then, proteins were precipitated overnight at −20°C. Precipitated proteins were centrifuged at 5000 × g for 30 minutes at 4°C. They were then washed three times in ice-cold acetone, air dried, and frozen at −80°C until use. For Acta1H40Y early-stage samples (both the mutant and WT), 40 μg of proteins was first separated onto an SDS-PAGE gel based on their different molecular weights and cut into 12 different bands. Gel pieces were then reduced with dithiothreitol and alkylated with iodoacetamide before being in-gel digested by trypsin (sequencing grade; Promega, Madison, WI) at room temperature overnight. After digestion, the peptides were extracted, pooled, and completely dried in a vacufuge, and peptides were resuspended in 20 μL of 50 mmol/L acetic acid for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. For the rest of three sets of samples, the protein pellets were weighted out and digested with trypsin (protein/enzyme, 100:1) in 50 mmol/L ammonium bicarbonate solution containing 0.1% RapiGest (Waters Corp., Milford, MA). Samples were reduced with dithiothreitol and alkylated with iodoacetamide before sequencing-grade trypsin was added. Digestion was performed overnight at 37°C. The following day, trifluoroacetic acid was added to a final concentration of 0.5% to the sample and incubated with the samples at 37°C for 30 minutes to precipitate RapiGest. The sample was then clarified at 15,000 × g for 5 minutes in a microcentrifuge, dried in a vacufuge, and resuspended in 40 μL of 50 mmol/L acetic acid. The concentration of the peptides was determined by nanodrop (absorbance 280 nm). Peptides obtained from Acta1H40Y early-stage and TgACTA1D286G samples were analyzed on a Thermo Fusion Orbitrap (Thermo Fisher, Coon Rapids, MN) instrument equipped with EASY-Spray Sources operated in positive ion mode. As described previously,21Wang H. Marrosu E. Brayson D. Wasala N.B. Johnson E.K. Scott C.S. Yue Y. Hau K.L. Trask A.J. Froehner S.C. Adams M.E. Zhang L. Duan D. Montanaro F. Proteomic analysis identifies key differences in the cardiac interactomes of dystrophin and micro-dystrophin.Hum Mol Genet. 2021; 30: 1321-1336Crossref PubMed Scopus (8) Google Scholar,22Bourdon A. Francois V. Zhang L. Lafoux A. Fraysse B. Toumaniantz G. Larcher T. Girard T. Ledevin M. Lebreton C. Hivonnait A. Creismeas A. Allais M. Marie B. Guguin J. Blouin V. Remy S. Anegon I. Huchet C. Malerba A. Kao B. Le Heron A. Moullier P. Dickson G. Popplewell L. Adjali O. Montanaro F. Le Guiner C. Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMD(mdx) rat model.Gene Ther. 2022; 29: 520-535Crossref PubMed Scopus (8) Google Scholar 1 hour LC gradient and preview mode data-dependent TopSpeed method was used for the analysis after peptide fractionation, either offline or online. Acta1H40Y late-stage samples were analyzed on a timsTOF Pro mass spectrometer (Bruker, Eden Prairie, MN) equipped with a CaptiveSpray source operated in positive ion mode using a 2-hour gradient (samples unfractionated). Briefly, samples were separated on a C18 reverse-phase column (1.6 μm particle size, 250 mm × 75 μm column size; IonOpticks, Fitzroy, VIC, Australia) using a Bruker nanoElute ultra–high-performance LC system. A total of 200 ng desalted peptide was injected for each analysis. Mobile phase A was 0.1% formic acid in water, and acetonitrile (with 0.1% formic acid) was used as mobile phase B. A flow rate of 0.4 μL/minute was used. Mobile phase B was increased from 2% to 17% over the first 60 minutes, then increased to 25% over the next 10 minutes, further increased to 37% over the next 10 minutes, and finally increased to 80% over 10 minutes and then held at 80% for 10 minutes. MS and MS/MS experiments were recorded over the m/z range 100 to 1700 and the ion mobility range (K0) of 0.6 to 1.6. Parallel accumulation serial fragmentation (PASEF) was used for all experiments, with the number of PASEF MS/MS scans set to 10. Active exclusion was applied, releasing after 0.4 minutes, with precursor reconsidered if current intensity/previous intensity was ≥4.0. Sequence information from the MS/MS data was then converted into a merged file (mgf) and analyzed using Mascot version 2.6.2 (Matrix Science, Boston, MA). Mascot was set up to search the most recent Uniprot_MOUSE_database. Trypsin was used as the digestion enzyme. Mascot was searched with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 10.0 parts per million. Carbamidomethyl of cysteine was specified in Mascot as a fixed modification. Deamidation of asparagine and glutamine and oxidation of methionine were specified as variable modifications. Scaffold version 5.0.1 (Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at >95.0% probability to achieve a false discovery rate of <1.0% by the Peptide Prophet algorithm23Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3980) Google Scholar with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at a false discovery rate of <1.0% and contained at least two identified peptides. Label-free quantitation was performed using the spectral count approach, in which the relative protein quantitation is measured by comparing the number of MS/MS spectra identified from the same protein in each of the multiple LC-MS/MS data sets.24Liu H. Sadygov R.G. Yates 3rd, J.R. A model for random sampling and estimation of relative protein abundance in shotgun proteomics.Anal Chem. 2004; 76: 4193-4201Crossref PubMed Scopus (2093) Google Scholar The t-test was performed by Scaffold to evaluate if the fold change for certain proteins is significant (P < 0.05). Principal component (PC) analysis, a dimensionality reduction technique, was used to visualize factors that contribute to each data set's variance. PC analyses were completed in R statistical computing software version 3.5.2 (https://www.r-project.org), performed as previously described.18Slick R.A. Tinklenberg J.A. Sutton J. Zhang L. Meng H. Beatka M.J. Vanden Avond M. Prom M.J. Ott E. Montanaro F. Heisner J. Toro R. Granzier H. Geurts A.M. Stowe D.F. Hill R.B. Lawlor M.W. Aberrations in energetic metabolism and stress-related pathways contribute to pathophysiology in the Neb conditional knockout mouse model of nemaline myopathy.Am J Pathol. 2023; 193: 1528-1547Abstract Full Text Full Text PDF Scopus (1) Google Scholar Briefly, the prcomp function, which uses singular value decomposition, was applied to each data set. Raw data were visually assessed using scree plots and then plotted using ggplot2.18Slick R.A. Tinklenberg J.A. Sutton J. Zhang L. Meng H. Beatka M.J. Vanden Avond M. Prom M.J. Ott E. Montanaro F. Heisner J. Toro R. Granzier H. Geurts A.M. Stowe D.F. Hill R.B. Lawlor M.W. Aberrations in energetic metabolism and stress-related pathways contribute to pathophysiology in the Neb conditional knockout mouse model of nemaline myopathy.Am J Pathol. 2023; 193: 1528-1547Abstract Full Text Full Text PDF Scopus (1) Google Scholar R code used for these analyses is provided in Supplemental Code 1. The Ingenuity Pathway Analysis (IPA) software package version 84978992 (Qiagen, Germantown, MD) was used to evaluate protein-set enrichment in the KI.Acta1H40Y versus WT.Acta1H40Y and TgACTA1D286G versus C57 data sets at early and late time points of disease. For each data set, expression (core) analyses were run using the measurement of expression fold change to calculate directionality z-scores.18Slick R.A. Tinklenberg J.A. Sutton J. Zhang L. Meng H. Beatka M.J. Vanden Avond M. Prom M.J. Ott E. Montanaro F. Heisner J. Toro R. Granzier H. Geurts A.M. Stowe D.F. Hill R.B. Lawlor M.W. Aberrations in energetic metabolism and stress-related pathways contribute to pathophysiology in the Neb conditional knockout mouse model of nemaline myopathy.Am J Pathol. 2023; 193: 1528-1547Abstract Full Text Full Text PDF Scopus (1) Google Scholar To validate changes in protein content observed in the proteomics data sets, Western blot analyses were performed on quadriceps tissue, as previously described,18Slick R.A. Tinklenberg J.A. Sutton J. Zhang L. Meng H. Beatka M.J. Vanden Avond M. Prom M.J. Ott E. Montanaro F. Heisner J. Toro R. Granzier H. Geurts A.M. Stowe D.F. Hill R.B. Lawlor M.W. Aberrations in energetic metabolism and stress-related pathways contribute to pathophysiology in the Neb conditional knockout mouse model of nemaline myopathy.Am J Pathol. 2023; 193: 1528-1547Abstract Full Text Full Text PDF Scopus (1) Google Scholar,25Lawlor M.W. Viola M.G. Meng H. Edelstein R.V. Liu F. Yan K. Luna E.J. Lerch-Gaggl A. Hoffmann R.G. Pierson C.R. Buj-Bello A. Lachey J.L. Pearsall S. Yang L. Hillard C.J. Beggs A.H. Differential muscle hypertrophy is associated with satellite cell numbers and Akt pathway activation following activin type IIB receptor inhibition in Mtm1 p.R69C mice.Am J Pathol. 2014; 184: 1831-1842Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar at 16 weeks of age. Polyvinylidene difluoride (PVDF) membranes were probed using antibodies listed in Table 1. Total protein was used for standardization, whereas the remaining targets were used to confirm signals seen in the proteomics dataset. Protein content was quantified using Image Lab Software version 6.1.0 build 7 (Bio-Rad Laboratories, Hercules, CA), and values were normalized to total protein visualized by Ponceau staining or stain-free blot activation. Total protein was used because of difficulty in finding a reliable loading control across all four genotypes.Table 1Primary Antibodies and Dilutions Used for Western Blot Analysis and Immunofluorescence StainingProteinAntibodyWestern blot analysis dilutionImmunofluorescence dilutionNRF2Abcam (Cambridge, UK) Ab1375501:7501:200KEAP1Abcam Ab2278281:10001:150NQO1Abcam Ab341731:10001:200SRFAbcam Ab531471:5001:75MRTF-AProteintech (Rosemont, IL) 21166-1-AP1:5001:200STARSProteintech 22673-1-AP1:10001:200VinculinCell Signaling (Danvers, MA) 13901S1:5000N/AEIF2S1Abcam Ab261971:5001:500ACTN3Creative Biolabs (Shirley, NY) 9B5N/A1:250ACTN3, alpha-actinin 3; EIF2, eukaryotic translation initiation factor 2; KEAP1, kelch-like ECH-associated protein 1; MRTF-A, myocardin-related transcription factor A; NRF2, nuclear factor erythroid 2–related factor 2; SRF, serum response factor; STARS, striated muscle activator of Rho signaling. Open table in a new tab ACTN3, alpha-actinin 3; EIF2, eukaryotic translation initiation factor 2; KEAP1, kelch-like ECH-associated protein 1; MRTF-A, myocardin-related transcription factor A; NRF2, nuclear factor erythroid 2–related factor 2; SRF, serum response factor; STARS, striated muscle activator of Rho signaling. Frozen quadriceps samples were sectioned transversely at 8 μm, and three tissue sections were mounted per slide. Tissue sections were stained with primary antibodies listed in Table 1 at 16 weeks of age to validate the proteomics data sets. Secondary antibodies include the following: goat anti-rabbit IgG Alexa Fluor 594 (Invitrogen, Waltham, MA; A11012, 1:400) and goat anti-mouse IgG1 Alexa Fluor 488 (Invitrogen; A21121; 1:400). Images were captured using a Leica SP8 Upright Confocal Microscope (Leica Microsystems, Buffalo Grove, IL). Immunohistochemistry was performed on the triceps of five male and five female mice of each genotype at 9 week
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