BAG3P215L/KO Mice as a Model of BAG3P209L Myofibrillar Myopathy
2020; Elsevier BV; Volume: 190; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2019.11.005
ISSN1525-2191
AutoresRebecca Robertson, Talita C. Conte, Marie-Josée Dicaire, Vladimir V. Rymar, Abbas F. Sadikot, Robert J. Bryson‐Richardson, Josée N. Lavoie, Erin O’Ferrall, Jason C. Young, Bernard Brais,
Tópico(s)Cardiovascular Effects of Exercise
ResumoBCL-2–associated athanogene 3 (BAG3) is a co-chaperone to heat shock proteins important in degrading misfolded proteins through chaperone-assisted selective autophagy. The recurrent dominant BAG3-P209L mutation results in a severe childhood-onset myofibrillar myopathy (MFM) associated with progressive muscle weakness, cardiomyopathy, and respiratory failure. Because a homozygous knock-in (KI) strain for the mP215L mutation homologous to the human P209L mutation did not have a gross phenotype, compound heterozygote knockout (KO) and KI mP215L mice were generated to establish whether further reduction in BAG3 expression would lead to a phenotype. The KI/KO mice have a significant decrease in voluntary movement compared with wild-type and KI/KI mice in the open field starting at 7 months. The KI/KI and KI/KO mice both have significantly smaller muscle fiber cross-sectional area. However, only the KI/KO mice have clear skeletal muscle histologic changes in MFM. As in patient muscle, there are increased levels of BAG3-interacting proteins, such as p62, heat shock protein B8, and αB-crystallin. The KI/KO mP215L strain is the first murine model of BAG3 myopathy that resembles the human skeletal muscle pathologic features. The results support the hypothesis that the pathologic development of MFM requires a significant decrease in BAG3 protein level and not only a gain of function caused by the dominant missense mutation. BCL-2–associated athanogene 3 (BAG3) is a co-chaperone to heat shock proteins important in degrading misfolded proteins through chaperone-assisted selective autophagy. The recurrent dominant BAG3-P209L mutation results in a severe childhood-onset myofibrillar myopathy (MFM) associated with progressive muscle weakness, cardiomyopathy, and respiratory failure. Because a homozygous knock-in (KI) strain for the mP215L mutation homologous to the human P209L mutation did not have a gross phenotype, compound heterozygote knockout (KO) and KI mP215L mice were generated to establish whether further reduction in BAG3 expression would lead to a phenotype. The KI/KO mice have a significant decrease in voluntary movement compared with wild-type and KI/KI mice in the open field starting at 7 months. The KI/KI and KI/KO mice both have significantly smaller muscle fiber cross-sectional area. However, only the KI/KO mice have clear skeletal muscle histologic changes in MFM. As in patient muscle, there are increased levels of BAG3-interacting proteins, such as p62, heat shock protein B8, and αB-crystallin. The KI/KO mP215L strain is the first murine model of BAG3 myopathy that resembles the human skeletal muscle pathologic features. The results support the hypothesis that the pathologic development of MFM requires a significant decrease in BAG3 protein level and not only a gain of function caused by the dominant missense mutation. Muscular dystrophy is a heterogeneous group of diseases characterized by progressive muscle weakness. Myofibrillar myopathies (MFMs) are a subset of muscular dystrophy that share pathologic features that may also present with cardiac muscle involvement and neuropathies. Symptoms and age at onset vary among the different MFMs, although most forms affect adults.1Selcen D. Ohno K. Engel A.G. Myofibrillar myopathy: clinical, morphological and genetic studies in 63 patients.Brain. 2004; 127: 439-451Crossref PubMed Scopus (232) Google Scholar Histologically and structurally, they all share similar features: Z-disk streaming and disruption, aggregation of Z-disk–associated proteins, abnormal fiber size,1Selcen D. Ohno K. Engel A.G. Myofibrillar myopathy: clinical, morphological and genetic studies in 63 patients.Brain. 2004; 127: 439-451Crossref PubMed Scopus (232) Google Scholar and mitochondrial abnormalities.2Jackson S. Schaefer J. Meinhardt M. Reichmann H. Mitochondrial abnormalities in the myofibrillar myopathies.Eur J Neurol. 2015; 22: 1429-1435Crossref PubMed Scopus (23) Google Scholar,3Vincent A.E. Grady J.P. Rocha M.C. Alston C.L. Rygiel K.A. Barresi R. Taylor R.W. Turnbull D.M. Mitochondrial dysfunction in myofibrillar myopathy.Neuromuscul Disord. 2016; 26: 691-701Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar In MFM, the broken-down filaments of the myofibers aggregate with membranous organelles and ectopic proteins.1Selcen D. Ohno K. Engel A.G. Myofibrillar myopathy: clinical, morphological and genetic studies in 63 patients.Brain. 2004; 127: 439-451Crossref PubMed Scopus (232) Google Scholar,4Olivé M. Odgerel Z. Martínez A. Poza J.J. Bragado F.G. Zabalza R.J. Jericó I. 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BAG3 also plays a role in the regulation of the stability of actin in conjunction with heat shock cognate protein 70 through interaction with the actin capping protein CapZ (a barbed-end actin capping protein from the Z line of skeletal muscle).8Hishiya A. Kitazawa T. Takayama S. BAG3 and Hsc70 interact with actin capping protein CapZ to maintain myofibrillar integrity under mechanical stress.Circ Res. 2010; 107: 1220-1231Crossref PubMed Scopus (106) Google Scholar Furthermore, it also plays an antiapoptotic role because of its association with B-cell lymphoma 2, an antiapoptotic protein localized to the mitochondria.9Hockenbery D. Nuñez G. Milliman C. Schreiber R. Korsmeyer S. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death.Nature. 1990; 348: 334-336Crossref PubMed Scopus (3535) Google Scholar The recurrent proline to leucine mutation in one of the Ile-Pro-Val motifs, P209L, results in a severe, childhood, autosomal-dominant MFM.10Selcen D. Muntoni F. Burton B.K. Pegoraro E. Sewry C. Bite A.V. Engel A.G. Mutation in BAG3 causes severe dominant childhood muscular dystrophy.Ann Neurol. 2009; 65: 83-89Crossref PubMed Scopus (274) Google Scholar Morphologically similar to other MFMs, these motifs display Z-disk streaming, aggregates of Z-disk proteins, abnormal fiber size, ragged red fibers (RRFs), abnormal mitochondrial morphologic features, and regenerating and splitting fibers.10Selcen D. Muntoni F. Burton B.K. Pegoraro E. Sewry C. Bite A.V. Engel A.G. Mutation in BAG3 causes severe dominant childhood muscular dystrophy.Ann Neurol. 2009; 65: 83-89Crossref PubMed Scopus (274) Google Scholar Clinically, affected patients begin experiencing symptoms in childhood, such as toe walking, that progress into more generalized muscle weakness, with spinal rigidity and a peripheral neuropathy in many patients. Respiratory insufficiency and cardiomyopathy usually develop by the second decade of life, often necessitating heart transplant and eventually leading to death.10Selcen D. Muntoni F. Burton B.K. Pegoraro E. Sewry C. Bite A.V. Engel A.G. Mutation in BAG3 causes severe dominant childhood muscular dystrophy.Ann Neurol. 2009; 65: 83-89Crossref PubMed Scopus (274) Google Scholar,11Odgerel Z. Sarkozy A. Lee H.-S. McKenna C. Rankin J. Straub V. Lochmüller H. Paola F. DtextquoterightAmico A. Bertini E. Bushby K. Goldfarb L.G. Inheritance patterns and phenotypic features of myofibrillar myopathy associated with a BAG3∼mutation.Neuromuscul Disord. 2010; 20: 438-442Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Because of the severity of the disease, the P209L mutation is usually de novo, with only one published case of it being inherited from a mosaic father.11Odgerel Z. Sarkozy A. Lee H.-S. McKenna C. Rankin J. Straub V. Lochmüller H. Paola F. DtextquoterightAmico A. Bertini E. Bushby K. Goldfarb L.G. Inheritance patterns and phenotypic features of myofibrillar myopathy associated with a BAG3∼mutation.Neuromuscul Disord. 2010; 20: 438-442Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar This finding makes this disease extremely rare, with only 19 published cases.10Selcen D. Muntoni F. Burton B.K. Pegoraro E. Sewry C. Bite A.V. Engel A.G. Mutation in BAG3 causes severe dominant childhood muscular dystrophy.Ann Neurol. 2009; 65: 83-89Crossref PubMed Scopus (274) Google Scholar, 11Odgerel Z. Sarkozy A. Lee H.-S. McKenna C. Rankin J. Straub V. Lochmüller H. Paola F. DtextquoterightAmico A. Bertini E. Bushby K. Goldfarb L.G. Inheritance patterns and phenotypic features of myofibrillar myopathy associated with a BAG3∼mutation.Neuromuscul Disord. 2010; 20: 438-442Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 12Lee H. Cherk S. Chan S. Wong S. Tong T. Ho W. Chan A. Lee K. Mak C. 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Dysregulated autophagy in restrictive cardiomyopathy due to Pro209Leu mutation in BAG3.Mol Genet Metab. 2018; 123: 388-399Crossref PubMed Scopus (43) Google Scholar One of the initial hypotheses concerning the mechanism of disease is that mutated BAG3 protein impairs autophagy,19Arimura T. Ishikawa T. Nunoda S. Kawai S. Kimura A. Dilated cardiomyopathy-associated BAG3 mutations impair Z-disc assembly and enhance sensitivity to apoptosis in cardiomyocytes.Hum Mutat. 2011; 32: 1481-1491Crossref PubMed Scopus (105) Google Scholar resulting in aggregates of misfolded proteins. This theory, however, has been disputed. Experiments in a zebrafish model found that suppression of autophagy is not enough to induce the formation of aggregates. Instead, only the expression of mutated BAG3 protein produces the aggregates, which also sequesters the wild-type (WT) protein, other Z-disk proteins, and autophagy-associated proteins. These results suggest that there is a toxic gain of function that leads to BAG3 insufficiency because the protein is now bound within aggregates and therefore unavailable to function.20Ruparelia A.A. Oorschot V. Vaz R. Ramm G. Bryson-Richardson R.J. Zebrafish models of BAG3∼myofibrillar myopathy suggest a toxic gain of function leading to BAG3 insufficiency.Acta Neuropathol. 2014; 128: 821-833Crossref PubMed Scopus (48) Google Scholar Indeed, the mutated protein stalled the HSP70 autophagy machinery because its activity as a nucleotide exchange factor is impaired. This finding leads to the formation of insoluble aggregates, which are composed of both the HSP70 system and other autophagic systems that accept the same clients,21Meister-Broekema M. Freilich R. Jagadeesan C. Rauch J.N. Bengoechea R. Motley W.W. Kuiper E.F.E. Minoia M. Furtado G.V. van Waarde M.A.W.H. Bird S.J. Rebelo A. Zuchner S. Pytel P. Scherer S.S. Morelli F.F. Carra S. Weihl C.C. Gestwicki S.B.J.E. Kampinga H.H. Myopathy associated BAG3 mutations lead to protein aggregation by stalling Hsp70 networks.Nat Commun. 2018; 9: 5342Crossref PubMed Scopus (41) Google Scholar preventing degradation and leading to their accumulation.21Meister-Broekema M. Freilich R. Jagadeesan C. Rauch J.N. Bengoechea R. Motley W.W. Kuiper E.F.E. Minoia M. Furtado G.V. van Waarde M.A.W.H. Bird S.J. Rebelo A. Zuchner S. Pytel P. Scherer S.S. Morelli F.F. Carra S. Weihl C.C. Gestwicki S.B.J.E. Kampinga H.H. Myopathy associated BAG3 mutations lead to protein aggregation by stalling Hsp70 networks.Nat Commun. 2018; 9: 5342Crossref PubMed Scopus (41) Google Scholar,22Guilbert S.M. Lambert H. Rodrigue M.-A. Fuchs M. Landry J. Lavoie J.N. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.FASEB J. 2018; 32: 3518-3535Crossref PubMed Scopus (37) Google Scholar Mice were generated with three different genotypes on C57Bl/6 background, carrying one [knock-in (KI)/ WT] or two copies of the P215L mutation analogous to the human P209L mutation (KI/KI), as well as mice with one copy of P215L and a knockout (KO) allele (KI/KO). A full characterization of heterozygote (KO/WT) mice was not completed because two other groups, both also using C57Bl/6 mice, independently reported that they did not develop a phenotype.23Youn D.Y. Lee D.H. Lim M.H. Yoon J.S. Lim J.H. Jung S.E. Yeum C.E. Park C.W. Youn H.J. Lee J.S. Lee S.B. Ikawa M. Okabe M. Tsujimoto Y. Lee J.H. Bis deficiency results in early lethality with metabolic deterioration and involution of spleen and thymus.Am J Physiol Endocrinol Metab. 2008; 295: E1349-E1357Crossref PubMed Scopus (27) Google Scholar,24Homma S. Iwasaki M. Shelton G.D. Engvall E. Reed J.C. Takayama S. BAG3 deficiency results in fulminant myopathy and early lethality.Am J Pathol. 2006; 169: 761-773Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar Extensive phenotypic, histologic, and molecular characterization of the KI/WT, KI/KI, and KI/KO mice was performed. KI/KO crosses were of the greatest interest because the KI/KI and KI/WT had little or no phenotype.25Fang X. Bogomolovas J. Zhou P.S. Mu Y. Ma X. Chen Z. Zhang L. Zhu M. Veevers J. Ouyang K. Chen J. P209L mutation in BAG3 does not cause cardiomyopathy in mice.Am J Physiol Heart Circ Physiol. 2019; 316: H392-H399Crossref PubMed Scopus (15) Google Scholar Further supporting the relevance of this cross are the results in zebrafish, which indicate that a reduction of protein quantity in addition to the P215L mutation may result in a muscle phenotype that is more severe than KI alone20Ruparelia A.A. Oorschot V. Vaz R. Ramm G. Bryson-Richardson R.J. Zebrafish models of BAG3∼myofibrillar myopathy suggest a toxic gain of function leading to BAG3 insufficiency.Acta Neuropathol. 2014; 128: 821-833Crossref PubMed Scopus (48) Google Scholar,26Ruparelia A.A. McKaige E.A. Williams C. Schulze K.E. Fuchs M. Oorschot V. Lacene E. Mirella M. Baxter E.C. Torrente Y. Ramm G. Stojkovic T. Lavoie J.N. Bryson-Richardson R.J. Metformin rescues muscle function in BAG3 myofibrillar myopathy models.bioRxiv. 2019; : 574806Google Scholar because the P209L mutation results in a lack of available and active BAG3.21Meister-Broekema M. Freilich R. Jagadeesan C. Rauch J.N. Bengoechea R. Motley W.W. Kuiper E.F.E. Minoia M. Furtado G.V. van Waarde M.A.W.H. Bird S.J. Rebelo A. Zuchner S. Pytel P. Scherer S.S. Morelli F.F. Carra S. Weihl C.C. Gestwicki S.B.J.E. Kampinga H.H. Myopathy associated BAG3 mutations lead to protein aggregation by stalling Hsp70 networks.Nat Commun. 2018; 9: 5342Crossref PubMed Scopus (41) Google Scholar In addition, a reduction in BAG3 levels has been noted in patients.14Kostera-Pruszczyk A. Suszek M. Płoski R. Franaszczyk M. Potulska-Chromik A. Pruszczyk P. Sadurska E. Karolczak J. Kamińska A.M. Rędowicz M.J. BAG3-related myopathy, polyneuropathy and cardiomyopathy with long QT syndrome.J Muscle Res Cell Motil. 2015; 36: 423-432Crossref PubMed Scopus (34) Google Scholar,27D'Avila F. Meregalli M. Lupoli S. Barcella M. Orro A. De Santis F. Sitzia C. Farini A. D'Ursi P. Erratico S. Cristofani R. Milanesi L. Braga D. Cusi D. Poletti A. Barlassina C. Torrente Y. Exome sequencing identifies variants in two genes encoding the LIM-proteins NRAP and FHL1 in an Italian patient with BAG3 myofibrillar myopathy.J Muscle Res Cell Motil. 2016; 37: 101-115Crossref PubMed Scopus (18) Google Scholar We hypothesized that reducing the protein quantity through a KI/KO cross would overcome the weak phenotype seen in KI/WT and KI/KI crosses in a manner that still reflected this dominant MFM on a molecular level. Indeed, KI/KO mice develop myopathic changes that are not present in the other strains. The BAG3-P219L KI/KO mice are the first model of P209L-BAG3 MFM with full-body expression of the mutant protein. Mice were generated by the Toronto Center for Phenogenomics using CRISPR-Cas9 on a C57Bl/6 background. The KI mice contain the change c.644_645CC>TG, which results in the P215L mutation. The KO mice have a 7-bp deletion, c.639_645delCATCCCC, causing a premature stop codon. The sequences were confirmed via Sanger sequencing using the primers 5′-GGTGTCTGGTGGCTATTTGG-3′ (forward) and 5′-ATCTGCAGAACCTCTCAGCC-3′ (reverse) (Supplemental Figure S1). The absence of protein was confirmed in the KO homozygote mice via Western blot using a polyclonal BAG3 antibody (LP11).22Guilbert S.M. Lambert H. Rodrigue M.-A. Fuchs M. Landry J. Lavoie J.N. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.FASEB J. 2018; 32: 3518-3535Crossref PubMed Scopus (37) Google Scholar Mouse gastrocnemius muscles were dissected and snap frozen in liquid nitrogen. Muscles were finely ground using a mortar and pestle, suspended in radioimmunoprecipitation assay buffer, and then sonicated. The resulting extract was spun at 10,000 × g for 5 minutes at 4°C and the supernatant collected. Protein quantification was performed using the DC colorimetric assay (Bio-Rad, Hercules, CA). Aliquots of 5, 20, and 40 μg were separated onto a NuPAGE 4% to 12% Bis-Tris gel (Life Technologies, Carlsbad, CA) and transferred onto a nitrocellulose membrane (Bio-Rad). The resulting blots were incubated with the following primary antibodies: BAG3 (LP11),22Guilbert S.M. Lambert H. Rodrigue M.-A. Fuchs M. Landry J. Lavoie J.N. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.FASEB J. 2018; 32: 3518-3535Crossref PubMed Scopus (37) Google Scholar p62 (catalog number 610832; BD Biosciences, San Jose, CA), HSPB8 (catalog number 3C12-H11; StressMarq, Victoria, BC), αB-crystallin (catalog number ADI-SPA-222; Enzo); HSPB1 (catalog number 5D12-A12; StressMarq), glyceraldehyde-3-phosphate dehydrogenase (catalog number GT239; GeneTex, Irvine, CA), HSP70 (clone C92F3A-5; Enzo Life Sciences, Farmingdale, NY), sarcomeric α-actinin (clone EA-53; Abcam, Cambridge, MA). This was followed by incubating membranes with a horseradish peroxidase secondary antibody and visualized using chemiluminscence (Intas Science Imaging, Göttingen, Germany). Bands were quantified using ImageJ software version 1.52 (NIH, Bethesda, MD; http://imagej.nih.gov/ij), and levels were normalized to glyceraldehyde-3-phosphate dehydrogenase and sarcomeric α-actinin. Cryostat sections of 10 μm were fixed with 4% paraformaldehyde for 10 minutes at room temperature and stained using the following stains: hematoxylin and eosin (H&E), Gomori trichrome, NADH, and immunohistochemical staining. For immunostaining, antigens were initially demasked in serial sections using target retrieval solution (catalog number S1699; Dako Agilent, Santa Clara, CA) at 95°C for 45 minutes. Peroxidases were inactivated using 0.6% hydrogen peroxide for 15 minutes, followed by avidin and biotin blocking (catalog number SP-2001; Vector Laboratories, Burlingame CA) for 15 minutes. Blocking and permeabilization were performed with a -buffered saline solution that contained 5% goat serum and 0.25% Triton X-100 for 1.5 hours. Primary antibodies were incubated overnight at 4°C. Biotinylated secondary antibodies (Vector Laboratories) were incubated for 1 hour. Complex A + B (catalog number PK-6100; Vector Laboratories) was incubated for 1 hour, followed by diaminobenzidine solution (catalog number SK-4100; Vector Laboratories) for 5 minutes. Nuclei were stained briefly using hematoxylin and then mounted with xylene mounting medium (catalog number 245-691; Protocol, Waltham MA). Primary antibodies consisted of the following: BAG3 (LP11),22Guilbert S.M. Lambert H. Rodrigue M.-A. Fuchs M. Landry J. Lavoie J.N. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.FASEB J. 2018; 32: 3518-3535Crossref PubMed Scopus (37) Google Scholar desmin (catalog number M0760; Dako), and filamin C (catalog number NBP1-89300; Novus Biologicals, Centennial CO). Counting of the RRFs, defined as fibers that have an irregularly thick or ragged contour, was performed using the Gomori trichrome–stained whole gastrocnemius sections. This counting was performed by an individual (R.R.) blind to the genotype associated with each section. The number of RRFs was quantified as a percentage of total fiber number. Cryostat cross-sections of 10 μm were fixed with 4% paraformaldehyde for 10 minutes at room temperature. They were blocked and permeabilized for 1 hour in a -buffered saline solution that contained 5% normal donkey serum, 0.25% Triton X-100, and 0.1 mol/L glycine. Cells were incubated overnight at 4°C with anti-CD45 primary antibody. The secondary antibody, mixed with 488 Alexa Fluor wheat germ agglutinin, was applied for 1 hour at room temperature, followed by Hoescht staining for 5 minutes. Slides were mounted with ProLongTM Gold antifade reagent (Thermo Fisher, Waltham, MA). Fiber-type staining immunolabelling was performed as previously described28Gouspillou G. Sgarioto N. Norris B. Barbat-Artigas S. Aubertin-Leheudre M. Morais J.A. Burelle Y. Taivassalo T. Hepple R.T. The relationship between muscle fiber type-specific PGC-1α content and mitochondrial content varies between rodent models and humans.PLoS One. 2014; 9: e103044Crossref PubMed Scopus (84) Google Scholar with the following primary antibody cocktail: anti–major histocompatibility complex (MHC) type I (BA-F8, 1:25), mouse IgG1 monoclonal anti-MHC type IIa (SC- 71, 1:25), mouse IgM monoclonal anti–type 2× MHC (6H1, 1:25), and a rabbit IgG polyclonal anti-laminin (catalog number L9393; Sigma-Aldrich, St. Louis, MO). The MHC monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, created by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the NIH and maintained at Department of Biology, The University of Iowa (Iowa City, IA; developed by Stefano Schiaffino). Male mice at 7 months of age, three per genotype used, were perfused with phosphate-buffered saline followed by 4% glutaraldehyde in 0.2 mol/L cacodylate buffer. The gastrocnemius muscle was dissected out, cut into 1 × 2-mm pieces, and left in the same glutaraldehyde mixture for 24 hours. Sections were postfixed in osmium tetroxide for 1 hour and embedded is epoxy resin. Sections were stained using uranyl acetate and lead citrate and then imaged using a FEI Tecnai G2 Spirit Twin 120 kV Cryo-TEM at the Facility of Electron Microscopy Research at McGill University. Motor behavior in mice was tested using rotarod, force transducers for grip strength, inverted grid, and open field. Fifteen male mice per genotype were used. Balance and coordination were tested using the rotarod. Mice were subjected to 3 days of training, gradually increasing the maximum Rpm through the training sessions and returning mice to the rotarod after falling unless exhaustion had been reached. The actual testing was composed of five sessions, with the speed increasing from 5 to 40 Rpm during that time. The time at which the mice fell from the rod was recorded, and adequate rest was given between rounds. For inverted grid, mice were placed on the grid, which was then inverted over a padded surface for a maximum of 5 minutes. The time at which the mice fell was recorded. Grip strength testing was performed using forelimbs only, with the peak force at the point of failure recorded for five trials. All values for rotarod, grip strength, and inverted grid were normalized to body weight, and these tests were repeated at 3, 6, and 9 months of age. For testing of spontaneous locomotion in open field, mice were allowed to acclimate to the testing room 30 minutes before testing while still in their home cages. They were then placed in a 50 × 50-cm field in a quiet, low-light room for 10 minutes and recorded using a ceiling-mounted camera and HVS Image Software version 2016.6 (HVS Image Software Ltd., Buckingham, UK). Testing was repeated at 5, 7, and 10 months of age. Individuals performing the testing were blind to genotype throughout the trials (R.R., M.-J.D.). Analyses that consisted of multiple genotypes were performed using two-way analysis of variance. If P < 0.05 was reached, post hoc testing (Dunnett multiple comparisons test) was performed to identify which group was different from WT. KI/WT, KI/KI, and KI/KO mice were clinically assessed using rotarod, inverted grid, grip strength, and open field tests to assess their strength, endurance, and movement. Inverted grid, rotarod, and weighing were performed at 3, 6, and 9 months, and grip strength and open field were performed at 4, 7, and 10 months. No significant difference among KI/WT, KI/KI, and WT mice was seen for any measure (Figure 1A). The KI/KO mice did not have any coordination, endurance, or strength deficit as measured by rotarod, inverted grid, or grip strength tests up to 9 months of age, and there were no differences in weight. However, the mice did have a significant reduction in voluntary movement (Figure 1B). Open field measurements revealed a significant decrease in movement, which became apparent at 7 months of age, moving a mean of 15.1 m compared with the 20.5 m by WT (total path and total entries P < 0.05) during a period of 10 minutes. This measurement decreased more at 10 months, moving 11.9 m compared with 17.85 m and moving 43% less of the time (total path P < 0.01, total entries and time moving P < 0.001). There was a decrease in overall movement and lower use of the field. The muscle of KI/KO mice has clear myopathic changes that are absent in KI/KI and KI/WT mice. A histologic examination of gastronomical muscle collected from 4-month–old male mice using H&E, Gomori trichrome, NADH, and immunohistochemical stains was conducted (Figure 2, A–C ). H&E staining of cross-sections of the gastrocnemius of 4-month–old mice revealed centralized nuclei, splitting fibers, and angular fibers in areas near connective tissue in the KI/KO animals (Figure 2D). These abnormalities did not increase markedly with age up to 14 months (Supplemental Figure S2). The RRFs were visible on Gomori trichrome (Figure 2G) staining. The RRF count in KI/KO muscle is significantly increased (5.5%) compared with WT muscle (1.1%) (P < 0.05) (Supplemental Figure S3, A and B). Although the KI/KI mice also had a higher level of RRFs (2.09%) than the WT mice, this finding was not significant. NADH staining also found moth-eaten fibers (Figure 2H), indicating myofibrillar disruption and significantly higher disruption in KI/KO (0.54%) compared with WT (0.08%) mice (P < 0.05), with a nonsignificant increase in KI/KI mice (0.29%) (Supplemental Figure S3C). Aggregates were seen in immunohistochemical stainings of the Z-disk proteins BAG3, desmin, and filamin C (Figure
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