Myofibrillar Instability Exacerbated by Acute Exercise in Filaminopathy
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Human Molecular Genetics, 2015, Vol. 24, No. 25 7207–7220 doi: 10.1093/hmg/ddv421 Advance Access Publication Date: 15 October 2015 Original Article ORIGINAL ARTICLE Myofibrillar instability exacerbated by acute exercise in filaminopathy Downloaded from Frédéric Chevessier1,†, Julia Schuld4,†, Zacharias Orfanos4,†, Anne-C. Plank2, Lucie Wolf1, Alexandra Maerkens5,6, Andreas Unger7, Ursula Schlötzer- 3 5 2 6 Schrehardt , Rudolf A. Kley , Stephan Von Hörsten , Katrin Marcus , http://hmg.oxfordjournals.org/ Wolfgang A. Linke7, Matthias Vorgerd5, Peter F. M. van der Ven4, Dieter O. Fürst4,* and Rolf Schröder1,* 1Institute of Neuropathology, 2Department for Experimental Therapy, Preclinical Experimental Animal Center and, 3Department of Ophthalmology, University Hospital Erlangen, Erlangen, Germany, 4Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn, Bonn, Germany, 5Department of Neurology, Neuromuscular Center Ruhrgebiet, University Hospital Bergmannsheil, 6Department of Functional Proteomics, at Erlangen Nuernberg University on August 15, 2016 Medizinisches Proteom-Center and 7Department of Cardiovascular Physiology, Ruhr-University Bochum, Bochum, Germany *To whom correspondence should be addressed at: Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn, Ulrich-Haberland-Str. 61a, D-53121 Bonn, Germany. Tel: +49-228-735301; Fax: +49-228-735302; Email: [email protected] (D.O.F.)/Institute of Neuropathology, University Hospital Erlangen, Schwabachanlage 6, D-91054 Erlangen, Germany. Tel: +49-9131-8534782; Fax: +49-9131-8526033; Email: [email protected] (R.S.) Abstract Filamin C (FLNC) mutations in humans cause myofibrillar myopathy (MFM) and cardiomyopathy, characterized by protein aggregation and myofibrillar degeneration. We generated the first patient-mimicking knock-in mouse harbouring the most common disease-causing filamin C mutation (p.W2710X). These heterozygous mice developed muscle weakness and myofibrillar instability, with formation of filamin C- and Xin-positive lesions streaming between Z-discs. These lesions, which are distinct from the classical MFM protein aggregates by their morphology and filamentous appearance, were greatly increased in number upon acute physical exercise in the mice. This pathology suggests that mutant filamin influences the mechanical stability of myofibrillar Z-discs, explaining the muscle weakness in mice and humans. Re-evaluation of biopsies from MFM- filaminopathy patients with different FLNC mutations revealed a similar, previously unreported lesion pathology, in addition to the classical protein aggregates, and suggested that structures previously interpreted as aggregates may be in part sarcomeric lesions. We postulate that these lesions define preclinical disease stages, preceding the formation of protein aggregates. Introduction with marked clinical and genetic heterogeneity due to mutations Filamin C-related protein aggregate myopathies belong to the in desmin, αB-crystallin, BAG-3, FHL1, myotilin, plectin and ZASP myofibrillar myopathies (MFMs), a numerically significant sub- (1,2). Filamins are large actin cross-linking proteins interacting group of hereditary and sporadic protein aggregate myopathies with a plethora of ligands of great functional diversity, indicating † These authors contributed equally. Received: August 31, 2015. Revised and Accepted: October 2, 2015 © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 7207 7208 | Human Molecular Genetics, 2015, Vol. 24, No. 25 both structural and signalling roles (3–6). Mutations in the ubiqui- the heterozygous genotype mirrors that of filaminopathy pa- tously expressed variants FLNA and FLNB cause a wide variety of tients, and direct comparison of the human and mouse filamino- congenital malformations, affecting brain, bone and other organs pathy pathology was a primary goal, we here focus on these mice (7–9). Heterozygous mutations of the human FLNC gene (located and refer to them as ‘mutant’ mice, and compare them with WT on chromosome 7q32; OMIM 102565) encoding the isoform main- littermates. ly expressed in striated muscles cause late-onset, autosomal- dominant and sporadic myopathies, and cardiomyopathies (10– 16). Filamin C-associated myopathies comprise three classical Expression levels of wild-type versus mutant filamin C disease manifestations: (i) protein aggregation myopathy affect- mRNA and protein ing skeletal and cardiac muscles with initial proximal weakness, To determine expression levels of WT and W2711X mutant Flnc (ii) distal myopathies due to haploinsufficiency or altered actin- mRNAs in our mice, we performed real-time reverse transcriptase- binding capacity without protein aggregation pathology and (iii) polymerase chain reaction (RT-PCR) (Fig. 2A) and semi-quantitative isolated hypertrophic cardiomyopathy without symptoms of multiplex RT-PCR (Supplementary Material, Fig. S1C) using RNA skeletal myopathy (11,16). We described the first pathological purified from different muscles and primer pairs amplifying mutation in filamin C (p.W2710X) in families from ethnically both WT and mutant Flnc cDNAs. Both indicated no significant diverse populations with a protein aggregation myopathy pheno- Downloaded from differences in total Flnc mRNA levels in mutant animals. Further- type, implying that codon 2710 is a mutational hotspot (10,13,17). more, experiments with a primer pair that enables discrimin- This mutation causes a deletion of the carboxy-terminal 16 ation of both genotypes due to the presence of the loxP site in amino acids, which abolishes the intrinsic dimer formation of exon48inmutantanimalsindicatedthatbothallelesareex- filamin C, making the mutant protein more prone to proteolysis pressed at similar levels in mutant animals (Fig. 2B). To exclude and aggregation in striated muscles (10,18). The aggregation that the lack of intact filamin C was compensated by increased pathology is characterized by sarcoplasmic and subsarcolemmal http://hmg.oxfordjournals.org/ expression of filamin A and/or B, mRNA expression levels of filamin C- and desmin-positive protein aggregates in conjunction Flna and Flnb were analysed. However, no significant differences with degenerative myofibrillar changes (1,2). between the two genotypes were detected (Supplementary Ma- Muscle biopsies from affected patients reflect only late stages terial, Fig. S1A and B). Concomitantly, Flnc was confirmed as the of the disease process and are only available in small amounts. main isoform in skeletal muscle (Supplementary Material, Moreover, biopsy material from preclinical, early and intermedi- Fig. S1D). ate disease stages is usually not accessible. With the purpose of Subsequently, we analysed filamin C protein expression in being able to perform pathophysiological and therapeutic inter- our mice with two different anti-filamin C antisera. One pan-fila- vention studies, we generated the first knock-in mouse model fi min C antiserum recognizing both WT and mutant variants for human MFM- laminopathy. In our mice, the expression of at Erlangen Nuernberg University on August 15, 2016 showed a highly similar protein level in both genotypes (further mutant W2710X filamin C (W2711X in mice) is controlled by confirmed by proteomic analysis, Supplementary Material, endogenous gene regulation sites, thus providing a patient- Table S1). A second antiserum raised against a peptide represent- mimicking genetic disease model. The subsequent analysis un- ing the carboxy-terminal 16 amino acids of filamin C that are ravelled a novel principle of pathogenesis, in which the mutant lacking in the mutant variant (that does not recognize filamins filamin C interferes with the mechanical stability and stress re- A and B and mutant filamin C, Supplementary Material, Fig. S7) sistance of myofibrillar Z-discs, subsequently leading to progres- showed that the WT filamin C level in the mutant animals was sive muscle damage. approximately half of that of the WT mice (Fig. 2C). This indicates that approximately half of the total filamin C in mutant animals Results represents mutant filamin C. Comparison of protein expression levels of the MFM aggregate markers BAG3, desmin, Hsp70 and fi Heterozygous W2711X lamin C knock-in mice: a XinB in WT and mutant animals by quantitative western blotting patient-mimicking pathogenetic model for human did not reveal significant differences (Fig. 2D). W2710X filaminopathy Heterozygous W2711X filamin C knock-in mice were generated Progressive muscle weakness in mutant mice using a gene-targeting strategy, which replaced the triplet ‘TGG’ by ‘TGA’, encoding a stop codon in exon 48, mimicking the most Analysis of the body weight of mutant and WT mice revealed no common MFM-filaminopathy mutation in man (10). Since mouse significant differences at any age between 2 and 12 months FLNc has an extra amino acid in the amino-terminal actin-bind- (Fig. 3A). No dysmorphology such as kyphosis or focal muscle at- ing domain, the codon 2711 in mice corresponds to codon 2710 in rophy was observed in mutant mice at any age (data not shown). the human ortholog. After homologous recombination and re- Animals were monitored repeatedly for motor function every 2 moval of the neomycin selection cassette by Flp-mediated exci- months up to 12 months by hanging-wire test to monitor sus- sion, the original gene structure is preserved and only one FRT tained limb strength (maximal time: 5 min; Fig. 3B) and direct site in intron 46