Zebrafish models of BAG3 myofibrillar myopathy suggest a toxic gain of function leading to BAG3 insufficiency

Abstract

Mutations in the co-chaperone Bcl2-associated athanogene 3 (BAG3) can cause myofibrillar myopathy (MFM), a childhood-onset progressive muscle disease, characterized by the formation of protein aggregates and myofibrillar disintegration. In contrast to other MFM-causing proteins, BAG3 has no direct structural role, but regulates autophagy and the degradation of misfolded proteins. To investigate the mechanism of disease in BAG3-related MFM, we expressed wild-type BAG3 or the dominant MFM-causing BAG3 (BAG3P209L) in zebrafish. Expression of the mutant protein results in the formation of aggregates that contain wild-type BAG3. Through the stimulation and inhibition of autophagy, we tested the prevailing hypothesis that impaired autophagic function is responsible for the formation of protein aggregates. Contrary to the existing theory, our studies reveal that inhibition of autophagy is not sufficient to induce protein aggregation. Expression of the mutant protein, however, did not induce myofibrillar disintegration and we therefore examined the effect of knocking down Bag3 function. Loss of Bag3 resulted in myofibrillar disintegration, but not in the formation of protein aggregates. Remarkably, BAG3P209L is able to rescue the myofibrillar disintegration phenotype, further demonstrating that its function is not impaired. Together, our knockdown and overexpression experiments identify a mechanism whereby BAG3P209L aggregates form, gradually reducing the pool of available BAG3, which eventually results in BAG3 insufficiency and myofibrillar disintegration. This mechanism is consistent with the childhood onset and progressive nature of MFM and suggests that reducing aggregation through enhanced degradation or inhibition of nucleation would be an effective therapy for this disease.

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References

  1. 1.

    Arimura T, Ishikawa T, Nunoda S et al (2011) Dilated cardiomyopathy-associated BAG3 mutations impair Z-disc assembly and enhance sensitivity to apoptosis in cardiomyocytes. Hum Mutat 32:1481–1491. doi:10.1002/humu.21603

    CAS  PubMed  Article  Google Scholar 

  2. 2.

    Arndt V, Dick N, Tawo R et al (2010) Chaperone-assisted selective autophagy is essential for muscle maintenance. Curr Biol 20:143–148. doi:10.1016/j.cub.2009.11.022

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Berghmans S, Butler P, Goldsmith P et al (2008) Zebrafish based assays for the assessment of cardiac, visual and gut function––potential safety screens for early drug discovery. J Pharmacol Toxicol Methods 58:59–68. doi:10.1016/j.vascn.2008.05.130

    CAS  PubMed  Article  Google Scholar 

  4. 4.

    Boglev Y, Badrock AP, Trotter AJ et al (2013) Autophagy induction is a Tor- and Tp53-independent cell survival response in a zebrafish model of disrupted ribosome biogenesis. PLoS Genet 9:e1003279. doi:10.1371/journal.pgen.1003279

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  5. 5.

    Bross P, Jespersen C, Jensen TG et al (1995) Effects of two mutations detected in medium chain acyl-CoA dehydrogenase (MCAD)-deficient patients on folding, oligomer assembly, and stability of MCAD enzyme. J Biol Chem 270:10284–10290

    CAS  PubMed  Article  Google Scholar 

  6. 6.

    Claeys KG, Ven PFM, Behin A et al (2009) Differential involvement of sarcomeric proteins in myofibrillar myopathies: a morphological and immunohistochemical study. Acta Neuropathol 117:293–307. doi:10.1007/s00401-008-0479-7

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Clemen CS, Herrmann H, Strelkov SV, Schröder R (2013) Desminopathies: pathology and mechanisms. Acta Neuropathol 125:47–75. doi:10.1007/s00401-012-1057-6

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  8. 8.

    Feldman AM, Begay RL, Knezevic T et al (2014) Decreased levels of BAG3 in a family with a rare variant and in idiopathic dilated cardiomyopathy. J Cell Physiol 229:1697–1702. doi:10.1002/jcp.24615

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Franaszczyk M, Bilinska ZT, Sobieszcza Ska-Ma Ek MG et al (2014) The BAG3 gene variants in Polish patients with dilated cardiomyopathy: four novel mutations and a genotype-phenotype correlation. J Transl Med 12:192. doi:10.1186/1479-5876-12-192

    PubMed Central  PubMed  Article  Google Scholar 

  10. 10.

    Fuchs M, Poirier DJ, Seguin SJ et al (2010) Identification of the key structural motifs involved in HspB8/HspB6-Bag3 interaction. Biochem J 425:245–255. doi:10.1042/BJ20090907

    CAS  Article  Google Scholar 

  11. 11.

    Fürst DO, Goldfarb LG, Kley RA et al (2013) Filamin C-related myopathies: pathology and mechanisms. Acta Neuropathol 125:33–46. doi:10.1007/s00401-012-1054-9

  12. 12.

    Gámez A, Pérez B, Ugarte M, Desviat LR (2000) Expression analysis of phenylketonuria mutations. Effect on folding and stability of the phenylalanine hydroxylase protein. J Biol Chem 275:29737–29742. doi:10.1074/jbc.M003231200

    PubMed  Article  Google Scholar 

  13. 13.

    Goldfarb LG, Park KY, Cervenáková L et al (1998) Missense mutations in desmin associated with familial cardiac and skeletal myopathy. Nat Genet 19:402–403. doi:10.1038/1300

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    Higashijima S, Okamoto H, Ueno N et al (1997) High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev Biol 192:289–299

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    Hishiya A, Salman MN, Carra S et al (2011) BAG3 directly interacts with mutated alphaB-crystallin to suppress its aggregation and toxicity. PLoS One 6:e16828. doi:10.1371/journal.pone.0016828

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  16. 16.

    Homma S, Iwasaki M, Shelton GD et al (2006) BAG3 deficiency results in fulminant myopathy and early lethality. Am J Pathol 169:761–773. doi:10.2353/ajpath.2006.060250

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  17. 17.

    Hunt L, Atherton J, McGaughran J (2014) Dilated cardiomyopathy––three brothers and a BAG3 mutation. Heart Lung Circ 23(Suppl 2):e11. doi:10.1016/j.hlc.2014.07.029

    Article  Google Scholar 

  18. 18.

    Kimmel CB, Ballard WW, Kimmel SR et al (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203:253–310. doi:10.1002/aja.1002030302

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Kley RA, Serdaroglu-Oflazer P, Leber Y et al (2012) Pathophysiology of protein aggregation and extended phenotyping in filaminopathy. Brain 135:2642–2660. doi:10.1093/brain/aws200

    PubMed Central  PubMed  Article  Google Scholar 

  20. 20.

    Kwan KM, Fujimoto E, Grabher C et al (2007) The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn 236:3088–3099. doi:10.1002/dvdy.21343

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Lee H, Cherk S, Chan S et al (2012) BAG3-related myofibrillar myopathy in a Chinese family. Clin Genet 81:394–398. doi:10.1111/j.1399-0004.2011.01659.x

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Lee JH, Takahashi T, Yasuhara N et al (1999) Bis, a Bcl-2-binding protein that synergizes with Bcl-2 in preventing cell death. Oncogene 18:6183–6190. doi:10.1038/sj.onc.1203043

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Lünemann JD, Schmidt J, Schmid D et al (2007) Beta-amyloid is a substrate of autophagy in sporadic inclusion body myositis. Ann Neurol 61:476–483. doi:10.1002/ana.21115

    PubMed  Article  Google Scholar 

  24. 24.

    Miles LB, Verkade H (2014) TA-cloning vectors for rapid and cheap cloning of zebrafish transgenesis constructs. Zebrafish 11:281–282. doi:10.1089/zeb.2013.0954

    PubMed  Article  Google Scholar 

  25. 25.

    Norton N, Li D, Rieder MJ et al (2011) Genome-wide studies of copy number variation and exome sequencing identify rare variants in BAG3 as a cause of dilated cardiomyopathy. Am J Hum Genet 88:273–282. doi:10.1016/j.ajhg.2011.01.016

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  26. 26.

    Odgerel Z, Sarkozy A, Lee H-S et al (2010) Inheritance patterns and phenotypic features of myofibrillar myopathy associated with a BAG3 mutation. Neuromuscul Disord 20:438–442. doi:10.1016/j.nmd.2010.05.004

    PubMed Central  PubMed  Article  Google Scholar 

  27. 27.

    Olivé M, Kley RA, Goldfarb LG (2013) Myofibrillar myopathies: new developments. Curr Opin Neurol 26:527–535. doi:10.1097/WCO.0b013e328364d6b1

    PubMed  Article  Google Scholar 

  28. 28.

    Pan T, Kondo S, Le W, Jankovic J (2008) The role of autophagy-lysosome pathway in neurodegeneration associated with Parkinson’s disease. Brain 131:1969–1978. doi:10.1093/brain/awm318

    PubMed  Article  Google Scholar 

  29. 29.

    Pfeffer G, Barresi R, Wilson IJ et al (2013) Titin founder mutation is a common cause of myofibrillar myopathy with early respiratory failure. J Neurol Neurosurg Psychiatr. doi:10.1136/jnnp-2012-304728

    PubMed  Google Scholar 

  30. 30.

    Ravikumar B, Vacher C, Berger Z et al (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 36:585–595. doi:10.1038/ng1362

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    Ruparelia AA, Zhao M, Currie PD, Bryson-Richardson RJ (2012) Characterization and investigation of zebrafish models of filamin-related myofibrillar myopathy. Hum Mol Genet. doi:10.1093/hmg/dds231

    PubMed  Google Scholar 

  32. 32.

    Ruparelia A, Vaz R, Bryson-Richardson R (2012) Myofibrillar myopathies and the Z-disk associated proteins. In: Cseri J (ed) Skeletal muscle––from myogenesis to clinical relations. InTech, Croatia, pp 317–358

    Google Scholar 

  33. 33.

    Schessl J, Zou Y, McGrath MJ et al (2008) Proteomic identification of FHL1 as the protein mutated in human reducing body myopathy. J Clin Invest 118:904–912. doi:10.1172/JCI34450

    CAS  PubMed Central  PubMed  Google Scholar 

  34. 34.

    Schröder R, Kunz WS, Rouan F et al (2002) Disorganization of the desmin cytoskeleton and mitochondrial dysfunction in plectin-related epidermolysis bullosa simplex with muscular dystrophy. J Neuropathol Exp Neurol 61:520–530

    PubMed  Google Scholar 

  35. 35.

    Selcen D, Engel AG (2004) Mutations in myotilin cause myofibrillar myopathy. Neurology 62:1363–1371

    CAS  PubMed  Article  Google Scholar 

  36. 36.

    Selcen D, Engel AG (2005) Mutations in ZASP define a novel form of muscular dystrophy in humans. Ann Neurol 57:269–276. doi:10.1002/ana.20376

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Selcen D, Engel AG (2011) Myofibrillar myopathies. Handb Clin Neurol 101:143–154. doi:10.1016/B978-0-08-045031-5.00011-6

    PubMed  Article  Google Scholar 

  38. 38.

    Selcen D, Muntoni F, Burton BK et al (2009) Mutation in BAG3 causes severe dominant childhood muscular dystrophy. Ann Neurol 65:83–89. doi:10.1002/ana.21553

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  39. 39.

    Takayama S, Xie Z, Reed JC (1999) An evolutionarily conserved family of Hsp70/Hsc70 molecular chaperone regulators. J Biol Chem 274:781–786

    CAS  PubMed  Article  Google Scholar 

  40. 40.

    Ulbricht A, Arndt V, Höhfeld J (2013) Chaperone-assisted proteostasis is essential for mechanotransduction in mammalian cells. Commun Integr Biol 6:e24925. doi:10.4161/cib.24925

    PubMed Central  PubMed  Article  Google Scholar 

  41. 41.

    Ulbricht A, Eppler FJ, Tapia VE et al (2013) Cellular Mechanotransduction Relies on Tension-Induced and Chaperone-Assisted Autophagy. Curr Biol. doi:10.1016/j.cub.2013.01.064

    PubMed  Google Scholar 

  42. 42.

    Vicart P, Caron A, Guicheney P et al (1998) A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy. Nat Genet 20:92–95. doi:10.1038/1765

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Vorgerd M, van der Ven PFM, Bruchertseifer V et al (2005) A mutation in the dimerization domain of filamin c causes a novel type of autosomal dominant myofibrillar myopathy. Am J Hum Genet 77:297–304. doi:10.1086/431959

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  44. 44.

    Wang J, Shaner N, Mittal B et al (2005) Dynamics of Z-band based proteins in developing skeletal muscle cells. Cell Motil Cytoskelet 61:34–48. doi:10.1002/cm.20063

    CAS  Article  Google Scholar 

  45. 45.

    Williams A, Sarkar S, Cuddon P et al (2008) Novel targets for Huntington’s disease in an mTOR-independent autophagy pathway. Nat Chem Biol 4:295–305. doi:10.1038/nchembio.79

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  46. 46.

    Winter L, Wiche G (2013) The many faces of plectin and plectinopathies: pathology and mechanisms. Acta Neuropathol 125:77–93. doi:10.1007/s00401-012-1026-0

    CAS  PubMed  Article  Google Scholar 

  47. 47.

    Wynn RM, Davie JR, Chuang JL et al (1998) Impaired assembly of E1 decarboxylase of the branched-chain alpha-ketoacid dehydrogenase complex in type IA maple syrup urine disease. J Biol Chem 273:13110–13118

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Dr. Peter van der Ven and Prof. Dieter Fürst for generously providing the full-length wild-type Filamin C construct, and the staff of the Monash FishCore facility for zebrafish care and maintenance. This work was supported by a Monash University Science Faculty Dean’s scholarship to A.A.R and an NHMRC discovery project Grant (#1010110). The contents of this manuscript are solely the responsibility of the authors and do not reflect the views of NHMRC.

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The authors declare no conflicts of interest.

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Correspondence to Robert J. Bryson-Richardson.

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Ruparelia, A.A., Oorschot, V., Vaz, R. et al. Zebrafish models of BAG3 myofibrillar myopathy suggest a toxic gain of function leading to BAG3 insufficiency. Acta Neuropathol 128, 821–833 (2014). https://doi.org/10.1007/s00401-014-1344-5

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Keywords

  • Myofibrillar myopathy
  • BAG3
  • Muscle
  • Zebrafish
  • Autophagy