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 C (FLNC) mutations in humans cause myofibrillar myopathy (MFM) and cardiomyopathy, characterized by 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 , αB-crystallin, BAG-3, FHL1, myotilin, and ZASP myofibrillar myopathies (MFMs), a numerically significant sub- (1,2). are large cross-linking 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]

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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 (located and refer to them as ‘mutant’ mice, and compare them with WT on 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 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 and two loxP sites in exon 46 and in the 3′ un- grip strength measurements of the forelimbs and all four paws translated region (UTR) encoding part of exon 48, respectively, re- (Fig. 3C). Remarkably, hanging-wire tests showed a decreased la- main. Southern blotting, polymerase chain reaction (PCR) tency to fall in mutant mice at all ages, becoming significant from genotyping and sequencing of genomic DNA confirmed correct the age of 10 months. However, statistically significant changes targeting (Fig. 1). in the forelimbs and four paw grip strength measurements be- Mating of heterozygous filamin C knock-in mice (10 inde- tween the WT and mutant mice were only noted at the age of 4 pendent crossings; n = 73 newborn mice) resulted in 26% wild months and at the ages of 4 and 8 months in the 4-paw (signifi- type (WT) (n = 19), 47% heterozygote (n = 34) and 27% homozygous cant interaction P < 0.0001) and forelimb grip strength testing mice (n = 20) indicating Mendelian inheritance, and suggesting (P < 0.05), respectively. Altogether, older mutant mice demon- no prenatal or perinatal death of W2711X knock-in mice. Since strated moderate yet significant signs of muscle weakness. Human Molecular Genetics, 2015, Vol. 24, No. 25 | 7209 Downloaded from http://hmg.oxfordjournals.org/ at Erlangen Nuernberg University on August 15, 2016

Figure 1. Gene-targeting strategy. (A) Schematic representation of filamin C protein. The W2710X (W2711X in mice) mutation is located in Ig-like domain 24 that is responsible for dimerization. Ig-like domain 20 is coloured differently since it contains an 82-amino acid-long insertion that is not present in filamin A or B. (B) Schematic diagram showing the strategy of the insertion of the W2711X mutation in Flnc in ES cells. Homologous crossing over leads to the replacement of exon 48 by a variant carrying the W2711X mutation and the insertion of the selection marker neomycin flanked by FRT sequences. Intron 46 and the part of exon 48 encoding the UTR contain loxP sites that would enable deletion of exon 47 and the protein-coding part of exon 48. Primers used for PCR (red arrows) and the probe used for Southern blot analysis (ext 5′ probe) are depicted together with the resulting amplicons and BglI genomic DNA fragments from the different alleles. (C) PCR analysis of tail DNA from the progeny of a mating between chimeric mice derived from the F1 breeding with Flp deleter mice to analyse the excision status of the neo cassette. WT, wild type; Mut, heterozygous mice; Mut/REC, mice carrying a WT allele and an allele with non-excised neo cassette. (D) Southern blot analysis of tail DNA from part of the progeny of a mating between mice carrying the heterozygous Flnc recombined and Flp deleter mice. DNAs were digested with BglI, transferred on nylon membrane and hybridized with the probe indicated in B.

Muscle pathology in mutant mice the sarcomere with scattered remnants of electron-dense mater- ial (Fig. 4E). This suggests a progressive and profound remodelling Staining of transverse cryosections from soleus (Supplementary of sarcomeric structures. Furthermore, areas with subsarcolem- Material, Fig. S2), quadriceps and gastrocnemius muscles (not mal accumulations of autophagic vacuoles indicating increased shown) of WT and mutant mice (3 and 8 months of age, 4 mice proteolytic activity were observed (Fig. 4F and G). per genotype) with haematoxylin and eosin (HE), modified Go- Though sedentary aged mutant animals did not develop mori trichrome, succinate dehydrogenase (SDH) and cytochrome muscle dystrophic changes, skeletal muscle specimens from oxidase (COX) essentially showed no obvious differences between 16-month-old mutant mice occasionally displayed degenerating WT and mutant animals. However, analysis of longitudinal sec- fibres (Supplementary Material, Fig. S3A) not seen in aged- tions of both genotypes by electron microscopy (EM) revealed matched WT mice. Note that the presence of ‘tubular aggregates’ many pathological alterations in the mutant mice that remained seen in our 16-month-old male mutant mice (Supplementary undetected by routine histochemical staining procedures. Al- Material, Fig. S3B) is a well-documented phenomenon in WT ready in 3-month-old mutant mice, fibres from soleus muscles dis- male mice (21,22) and is not linked to the pathology under played Z-disc streaming, i.e. sarcomeric disruptions characterized investigation. by electron-dense material bridging adjacent Z-discs (Fig. 4A), re- sembling the sarcomeric ‘lesions’ typically seen after eccentric exercise (19,20). In other areas, myofibrillar degeneration with Myofibrillar lesion formation in mutant mice and disassembled Z-discs (Fig. 4B, asterisks) associated with fused, filaminopathy patients enlarged mitochondria was seen (Fig. 4B, arrowheads). Myofibrils at different stages of degeneration were observed, ranging from Although we did not find typical signs of MFM-related protein mild Z-disc pathology (Fig. 4C) over disassembly of the thin– aggregation by histochemical staining, immunohistochemistry thick filament systems (Fig. 4D), to complete disintegration of revealed focal Z-disc streaming in longitudinal sections, 7210 | Human Molecular Genetics, 2015, Vol. 24, No. 25 Downloaded from http://hmg.oxfordjournals.org/ at Erlangen Nuernberg University on August 15, 2016

Figure 3. Body weight and clinical phenotyping. (A) Body weight of mice carrying the heterozygous recombinant W2711X allele (Mut) and their WT littermates at different ages between 2 and 12 months. No significant differences in body weight are observed between WT and mutant mice at any age. (B) Hanging-wire tests performed at different ages show a statistically significant decreased latency to fall in mutant mice starting at the age of 10 months (10 months, P = 0.0177; 12 months, P = 0.0038). (C) Forelimbs and four paw grip strength measurements of WT and mutant mice aged between 2 and 12 months. Statistically significant changes were only noted at the age of 4 months (P < 0.0001) and at the ages of 4 (P = 0.0362) and 8 (P = 0.0241) months in the fi Figure 2. Expression of lamin C mRNA and protein levels in soleus muscles of WT 4-paw and forelimb grip strength testing, respectively (WT: n =5;Mut: n =7; and mutant (Mut) mice. (A) Real-time PCR (with Gapdh as internal normalization error bars represent ±standard deviation). control, n = 5 per genotype) indicates no significant differences in total Flnc mRNA levels between WT and mutant mice. (B) RT-PCR analysis shows that both the WT and recombinant W2711X knock-in allele (KI) are expressed in mutant animals particularly in solei of mutant mice. Z-disc streaming was char- (amplicons derived from the recombined locus are longer since they include the acterized by intensified filamin C staining bridging two or more loxP site in exon 48). (C) Analysis of filamin C protein expression in WT and neighbouring Z-discs (Fig. 5Ai), resembling previously described mutant mice muscles using an antiserum recognizing both filamin C variants (FlnC total) and an antiserum specificforWTfilamin C (FlnC WT). Total filamin C sarcomeric lesions after eccentric exercise (23). They correspond levels are not altered compared with WT animals, whereas WT filamin C levels are to sarcomeric lesions seen by EM (Fig. 4A). Occasionally, these approximately half of the total level, indicating that the other half represents structures spanned very large areas, across many myofibrils and mutant filamin C. (D) Comparison of the protein expression levels of the MFM covering almost the entire fibre width (Fig. 5Aii). To differentiate aggregate markers BAG3, desmin, Hsp70 and XinB in WT (red line) and mutant between the extent of those structures, we refer to them here as animals. None of the analysed proteins is expressed at significantly altered levels microlesions (spanning up to five sarcomeres) and macrolesions in mutant animals. GAPDH was used as normalization control. Human Molecular Genetics, 2015, Vol. 24, No. 25 | 7211 Downloaded from http://hmg.oxfordjournals.org/ at Erlangen Nuernberg University on August 15, 2016

Figure 4. Ultrastructural analysis of the soleus muscle of mutant mice. (A and B) Typical structural anomalies in the mutant mouse muscles showing sarcomeric lesions (Z-disc streaming) with electron-dense material connecting adjacent Z-discs (arrows), autophagic vacuoles (white arrowhead) and the progressive stages of myofibrillar degeneration (asterisks; white asterisk more pronounced) including disassembled Z-discs and enlarged mitochondria (arrowheads). (C–E) Different stages of Z-disc disintegration (arrows), culminating in complete myofibril degeneration. Note enlarged mitochondria (arrowheads in D and E). (D) Only remnants of the Z-disc and the intermyofibrillar (arrow) are preserved in advanced stages. (E) Complete dissolution of the Z-disc structure in the late stages of the sarcomeric deterioration. (F and G) Frequent observation of subsarcolemmal depositions including enlarged mitochondria (black arrowheads) and autophagic vesicles (white arrowheads). The boxed area in F is shown enlarged in G. Scale bars: (A) 2.3 µm and (B–F) 250 nm.

(more than five sarcomeres and across multiple myofibrils). different mutations in filamin C [W2710X, V930_T933del and a re- Macrolesions were frequently seen in close association with cently identified mutation in domain 24, see Patient 6 in (24)]. In- blood vessels (Supplementary Material, Fig. S4). In transverse sec- deed, we observed micro- and macrolesions in all three samples tions, lesions were seen as high-intensity filamin C-positive areas (Fig. 5; Supplementary Material, Fig. S6), indicating that the lesion of variable size (Fig. 5Aiii). Furthermore, COX and SDH stains of pathology is a common phenomenon in human and mouse transverse serial sections suggested the absence of mitochondria filamin C-associated pathology. However, MFM-related protein in those regions (Supplementary Material, Fig. S5). Even though aggregates were only observed in patient (Fig. 5B and C; Supple- such lesions were not found in every fibre when examining a sec- mentary Material, Fig. S6) and not in mutant mouse skeletal mus- tion, they were, however, seen in each preparation. cle tissue. Sarcomeric lesions can be distinguished from We subsequently looked for the presence of such micro- and amorphous and rounded aggregates via their filamentous ap- macrolesions in longitudinal sections of the diagnostic muscle pearance and their association with the myofibrils in a striated biopsies of human filaminopathy patients carrying three manner (see Supplementary Material, Fig. S8). Interestingly, in 7212 | Human Molecular Genetics, 2015, Vol. 24, No. 25 Downloaded from http://hmg.oxfordjournals.org/ at Erlangen Nuernberg University on August 15, 2016

Figure 5. Soleus muscles of the mutant mouse show sarcomeric lesions similar to those found in human MFM-filaminopathy. (A) (i) Filamin C staining of mutant mouse muscle fibres reveals small filamin C-positive ‘microlesions’ (µ), spanning 1–5 sarcomeres, (ii) ‘macrolesions’ (M) spanning larger areas are also seen and (iii) both structures are identifiable in transverse sections. (B)Musclefibres of a human W2710X filaminopathy patient contain similar micro- and macrolesions flanked by large filamin C-positive protein aggregates (Ag) appearing rounded and amorphous, whereas lesions have a striated appearance. Boxed areas in (B) are shown enlarged below the upper figures. Lesions (µ, M) and protein aggregates (Ag) are positive for filamin C and Xin, known markers of sarcomeric lesions. (C) Aggregates (Ag) in W2710X patient muscle fibres contain filamin C, Xin and desmin. Note that desmin is accentuated in the periphery of the aggregates. Scale bars: 10 µm. patients, both structures were positive for the sarcomeric lesion- Acute strenuous exercise exacerbates the formation of markers filamin C, desmin, Xin (Fig. 5C; Supplementary Material, lesions in limb muscles and diaphragm of mutant mice Fig. S7) and myotilin (Supplementary Material, Fig. S7). Regarding We then examined the effects of a single bout of acute strenuous desmin, it is noteworthy that its immunoreactivity was accentu- treadmill exercise on the formation of lesions in soleus muscles of ated at the periphery of individual aggregates in the W2710X pa- mutant and WT mice. For quantification of lesion formation, tient (Fig. 5C). transverse sections were stained to visualize the effect across Human Molecular Genetics, 2015, Vol. 24, No. 25 | 7213

the whole muscle. Large areas positive for the lesion marker Xin, number of microlesions, the effect in mutant mice was consider- corresponding to macrolesions, were present in muscles of exer- ably more pronounced. In parallel, also the number of exercise- cised mutant animals but not in exercised WT control muscles induced macrolesions was dramatically increased in mutant (Fig. 6A). Micro- and macrolesions were quantified (Fig. 6B). mice (Fig. 6). Altogether, these results show that soleus muscles Even though some microlesions were occasionally found in ofmutantmicearemorepronetomyofibrillar instability and non-exercised WT solei, their number was significantly higher damage than WT mice, and that this is exacerbated during in the non-exercised mutant mice. Moreover, large macrolesions acute intensive exercise. seen already in sedentary mutant mice were not observed in sed- As the macrolesions were generally located near the sarco- entary WT mice. The chosen acute strenuous exercise protocol lemma and could be mistaken for the well-known MFM-related had a dramatic effect on the occurrence of both lesion types. protein aggregates, we further characterized their pattern of im- Whereas running WT mice showed a moderate increase in the munoreactivity with established lesion and sarcolemma marker Downloaded from http://hmg.oxfordjournals.org/ at Erlangen Nuernberg University on August 15, 2016

Figure 6. High prevalence of sarcomeric lesions in the soleus muscles of mutant animals, exacerbated by treadmill exercise. (A) Examples of transverse sections through soleus muscles after exercise; individual fibres demarcated by staining for the caveolin 3 (Cav3, red), lesions detected by the lesion marker Xin (green). Note the high prevalence of fibres with Xin-positive areas in the mutant. (B) Prevalence of micro- and macrolesions in the WT and mutant animals before and after exercise, expressed as the number of lesion-positive fibres per section. Even before exercise, significantly higher numbers of both micro- and macrolesions are seen in mutant muscles compared with WT muscles. After exercise, the number of both structures increased in both genotypes, with a significantly higher number of macrolesions in the mutant animals. (C) Xin-positive macrolesions as seen in longitudinal sections are most commonly associated with the fibre membrane. (D) Membrane marker -G (Ank G) is seen weaker or absent in positions where macrolesions meet the membrane. (E) Membrane-bound macrolesions detected in transverse sections are positive for BAG3 and HSP27 but negative for desmin. Scale bars: (A) 200 µm and (C–E) 10 µm. 7214 | Human Molecular Genetics, 2015, Vol. 24, No. 25

proteins. Like filamin C, the lesion marker Xin strongly stained fully integrated, automated intra-home-cage monitoring system. macrolesions (Fig. 6C). The well-known costameric filamin C-bind- Both mutant and WT mice showed a significant reduction in total ing partners ankyrin-G (Fig. 6D) and caveolin-3 (data not shown) locomotor activity after exercise compared with corresponding were markedly reduced or even absent in sarcolemmal areas adja- dark-phase activity levels measured before exercise; however, cent to macrolesions. Transverse sections through macrolesions the effect was stronger in mutant mice (Fig. 8A). Even more pro- also showed immunoreactivity with the autophagy marker BAG3 nounced, after acute exercise rearing activity (Z plane activity and the small heat shock protein HSP27 (Fig. 6E), both established counts) was significantly reduced only in mutant mice (P = 0.008), protein aggregate components. This suggests that structures typ- while that of WT mice remained largely unchanged (Fig. 8B). This ically identified as aggregates in such sections may in fact be may reflect an increased post-exhaustion effect in mutant mice. macrolesions. Although morphologically distinct, both macrole- The respiratory exchange ratio (RER) is defined as the ratio of sions and protein aggregates show increased immunoreactivities CO2 produced and O2 consumed. Values approaching 0.70 are in- for the same markers. Surprisingly, the classical MFM-marker des- dicative of fat being the predominant source of energy, whereas min was not enriched in the macrolesions in the mouse (Fig. 6E), those around 1.00 refer to a more carbohydrate-based metabol- even though both lesions and aggregates in the patient were des- ism. The RER of all mice tested followed a circadian pattern min positive either in the centre or the periphery of these struc- with higher values during activity (dark phases) than at rest

tures (Fig. 5C; Supplementary Material, Fig. S7). (light phases) (Fig. 8C). However, compared with WT mice, RER Downloaded from Since respiratory insufficiency is one of the most severe com- values of mutant mice were slightly lower at baseline conditions plications in MFM-filaminopathy patients, we also analysed the (Fig. 8C). Food consumption of mutant animals was also slightly appearance of lesions in the diaphragm of exercised mice. While reduced (Supplementary Material, Fig. S9A). After acute exercise, staining for filamin C did not reveal any lesions in the diaphragms mean dark-phase RER values were significantly reduced in mu- fi of WT mice (Fig. 7B and D), numerous lesions were seen in the - tant mice (Fig. 8D and E), which is due to a lower mean CO2 pro- bres of mutant animals (Fig. 7A and C), indicating that those mus- duction, while O2 consumption remained rather constant http://hmg.oxfordjournals.org/ cles are especially vulnerable to the strain induced by exercise. (Supplementary Material, Fig. S9B and C). These data are congru- ent with reduced food consumption during the dark phase after acute exercise (Supplementary Material, Fig. S9A), as well as the Effects of acute exercise on physical activity and diminished post-exercise activity levels observed in both geno- respiratory exchange ratios in mutant mice types (Fig. 8A). However, the altered RER values in mutant ani- The drinking and feeding behaviour, metabolic performance and mals could also be aggravated by insufficient respiratory home-cage activity in X + Y as well as Z planes of mutant and WT biomechanics inflicted by exercise-induced multiple lesion for- mice were monitored before and after treadmill exercise using a mation in the fibres of the diaphragm (Fig. 7). at Erlangen Nuernberg University on August 15, 2016

Figure 7. Sarcomeric lesions in the diaphragm of exercised mutant mice. Immunolocalization of filamin C on longitudinal (A and B) and transversal (C and D) sections of diaphragms of exercised mutant (A and C) and WT (B and D) animals. Note the presence of large numbers of micro- and macrolesions only in the muscle fibres of the mutant animals. Scale bar: 40 µm. Human Molecular Genetics, 2015, Vol. 24, No. 25 | 7215 Downloaded from http://hmg.oxfordjournals.org/ at Erlangen Nuernberg University on August 15, 2016

Figure 8. Physical activity and respiration of exercised WT and mutant mice. Total XY-plane (A) and Z-plane (B) activity counts per dark phase before and after acute exercise of mutant (Mut) and WT mice (n = 4, mean with SEM). (A) Both groups show a significant reduction in total locomotor activity after acute treadmill exercise compared with baseline activity levels (Mut: P < 0.001; WT: P = 0.013). (B) Rearing activity is significantly reduced in mutant (P = 0.002) but not in WT animals. (C–E) Circadian rhythmicity of RER values (average per min, 20 min bins) before (C) and after (D) acute exercise. RER values of heterozygous mice are slightly lower at baseline conditions compared with WT RER values and significantly reduced (P = 0.02) in the dark phase after acute exercise (E; mean with SEM).

Discussion Our heterozygous W2711X Flnc knock-in mice are the first pa- FLNC mutations cause a late-onset protein aggregate myopathy tient-mimicking animal model for this disease. To ensure that and cardiomyopathy leading to progressive muscle weakness, these mice indeed reflect the situation in patients, we confirmed often causing premature death (10–12,17,25). The lack of cell that the mutant allele is expressed without elimination of mu- and animal models for the disease and of patient biopsy material tant mRNA by nonsense-mediated decay. WT and mutant fila- from preclinical disease stages has impeded deciphering the mo- min C proteins were expressed in similar quantities, with the lecular pathogenesis behind the onset and progression of the total filamin C amount comparable with that of WT mice. The lat- disease. To overcome these limitations, we generated an MFM- ter finding was further corroborated by proteomic analysis. This filaminopathy mouse model, harbouring the mouse ortholog of rules out that our gene-targeting strategy leads to haploinsuffi- the most prevalent human p.W2710X filamin C mutation (10). ciency, or otherwise reduced levels of filamin C, as has been 7216 | Human Molecular Genetics, 2015, Vol. 24, No. 25

reported in other filamin C-related mouse (26)orfish models The presence of lesions and aggregates in single fibres also (27,28). provides a connection between both pathologies. The presence Histological analyses of skeletal muscle from our sedentary of lesions even in sedentary mutant mice suggests that they heterozygous mice did not show any overt alterations up to the may form in patients long before the presentation of a clinical age of 8 months. However, at the ultrastructural level, abnormal- phenotype and could be utilized as a preclinical predictor of my- ities such as enlarged mitochondria and autophagic vacuoles opathy. It has been previously postulated that macrolesions seen were already observed in 3-month-old mutant mice. Further- after eccentric exercise may escalate progressively from microle- more, even though no MFM-associated protein aggregates were sions (43). Based on our findings in mice and men, it is tempting seen, different stages of myofibrillar degeneration starting at Z- to speculate that macrolesions eventually give rise to amorphous discs, and myofibrillar lesions were observed. These lesions are protein aggregates in MFM-filaminopathy patients. Diagnostic areas of focal disruption resembling those implicated in post-ex- specimens of patients showing massive aggregation pathology ercise remodelling (29,30). They appear as electron-dense mater- are only taken after the disease has progressed to a symptomatic ial ‘streaming’ between adjacent Z-discs and have also been phenotype, usually at an age that can hardly be recapitulated in described as Z-disc streaming (19,20,29,31). Lesions border on a mice. Indeed, large protein aggregates typical for MFM-filamino- Z-disc and can span as little as a single sarcomere or extend pathy patients were not observed in our mutant mice. We, there-

across multiple sarcomeres and include several laterally neigh- fore, suggest that protein aggregation represents a late stage of Downloaded from bouring myofibrils. They display strongly enhanced staining for the disease and is a consequence of the increasing formation of filamin C, its binding partners Xin, myotilin, aciculin and actin, lesions and/or progressive difficulties to cope with the high de- as well as desmin (23,30,32–34). Even though these structures mand for repair. Currently, the most accepted assumption is are also found in healthy, unexercised individuals (35), their that during early life, damaged filamin C (WT as well as mutant) prevalence increases after eccentric exercise (20,36–39). Here, is continuously removed from muscle fibres, mostly by chaper- we show that this prevalence is significantly higher in our mu- one-assisted selective autophagy (CASA) and the ubiquitin– http://hmg.oxfordjournals.org/ tant mice, even when those were sedentary. Note that in our proteasome system (44,45). The high level of mutant filamin C mice (in contrast to the patients), macrolesions were negative in our W2711X mice, however, indicates that even in early life for desmin. It has been documented, however, that young lesions these processes are not sufficiently active to efficiently remove are desmin negative, only becoming desmin positive later, after misfolded, mutant filamin C. In addition, it suggests that filami- remodelling has been initiated (23,32), and sampling was per- nopathy patients express equally large quantities of mutant fila- formed very soon after the exercise experiments in our mice. min C in their muscles. Considering the absence of protein We validated the lesion pathology in human MFM-filamino- aggregates in the mice and the many unaffected Z-disc- and pathies by analysing longitudinal sections from diagnostic mus- sarcolemmal protein-binding sites in mutant filamin C, we as-

cle biopsy specimens from patients carrying different MFM- sume that it is localized together with the normal protein at at Erlangen Nuernberg University on August 15, 2016 causing FLNC mutations. This revealed—in addition to the char- Z-discs and sarcolemma. In contrast to overexpressed mutant acteristic amorphous protein aggregates—a large number of filamin C in cultured cells (17,18), it does not seem to spontan- micro- and macrolesions. Indeed, structures previously charac- eously aggregate. Since we cannot specifically immunolocalize terized as aggregates or early mini-aggregates in transverse mus- the mutant protein, we cannot rule out that it remains soluble cle sections are at least in part macrolesions and microlesions, until its degradation. In any case, its expression does not lead respectively (24). Since macrolesions in our mice appeared nega- to clinically pronounced muscle weakness in the first decades tive for histological mitochondrial stains, a characteristic of of patient ’s life. The reason for the development of aggregates MFM-associated ‘rubbed-out areas’ (40,41), we cannot exclude only in later life remains to be determined, but is probably due that in patients some of these areas correspond to macrolesions to overstrain of the ubiquitin proteasome and autophagy path- or aggregates. Thus, our data in mice and men demonstrate that ways that show reduced activity in later life [reviewed in (46,47)]. lesions are a typical sign of MFM-filaminopathy (and possibly The Z-disc pathology and lesion formation in mutant animals other MFMs) in early and late stages of the disease. In the routine can be explained on the basis of the proposed functions of filamin diagnostic work-up of human muscle biopsies, the standard is C. At Z-discs, it interacts with multiple partners such as myotilin, interpretation of transverse sections; however, differentiation aciculin, myopodin and FATZ/calsarcin/myozenin (34,48–53). between lesions and aggregates is not possible by that approach, These interactions and its upregulation early during myocyte dif- especially as the protein markers commonly used for detection ferentiation imply a role for filamin C in myofibril assembly and are present in both structures. maintenance (26,48,54). The human W2710X mutant filamin C Intra-home-cage monitoring including indirect calorimetry lacks the last 16 amino acids of Ig-like domain 24 and is unable suggested that the respiration of mutant animals is affected post- to dimerize in vitro (10,18,55). This probably interferes with its exercise. Formation of sarcomeric lesions is considered to cause cross-linking and stabilizing function at Z-discs, thereby causing post-exercise muscle weakness, as they represent disruptions of the observed Z-disc pathology. At the sarcolemma, filamin C in- the force-generating myofibrils (39,42). Therefore, the lesion path- teracts with γ-andδ- sarcoglycans, ankyrin-G and ponsin ology observed in diaphragm and skeletal muscles of our mutant (54,56,57). We previously demonstrated reduced binding of the mice may explain both respiratory insufficiency and muscle weak- truncated domain to both sarcoglycans (18), implying that it ness experienced by MFM-filaminopathy patients. In contrast to le- can no longer associate with the -associated glycopro- sions, protein aggregates in patients are cytosolic and largely found tein complex. This might destabilize this complex and increase flanking intact myofibrils. We, therefore, propose that the lesion the prevalence of local membrane damage. Indeed, we found re- pathology, rather than the presence of cytoplasmic aggregates, is duced ankyrin-G and caveolin 3-staining at the sarcolemma the leading cause of muscle weakness in these patients. Assuming flanking macrolesions. Notably, these macrolesions frequently that the occurrence of lesions is a normal process in striated occurred in the vicinity of blood vessels, adding local mechanical muscles, their increased occurrence in our mutant mice and pressure to the flanking muscle fibres, further indicating their in- MFM-filaminopathy patients indicates either a decreased stability creased mechanical susceptibility to damage. Taken together, of myofibrils or a delay in repair processes. the expression of mutant filamin C induces a dual pathology at Human Molecular Genetics, 2015, Vol. 24, No. 25 | 7217

the level of Z-discs and the sarcolemma, presumably due to its Science Associations (FELASA). Mice were handled in accordance impaired stabilizing function. with the German Animal Welfare Act and the German Regulation Beyond novel insight into the molecular pathogenesis of fila- for the protection of animals used for experimental purposes or minopathies, our work has implications for counselling of af- other scientific purposes. For experiments, mice were euthanized fected patients. Acute strenuous exercise leads to a dramatic by a lethal dose of isoflurane followed by cervical dislocation. increase in myofibrillar lesion pathology. Translated into the fi human disease context, our ndings strongly imply that MFM- Genotyping of mice filaminopathy patients are more susceptible to acute exercise- induced myofibrillar damage, which is likely to contribute to Tail DNA was obtained using DNeasy Blood & Tissue Kit (Qiagen) the progressive muscle weakness. However, the important ques- according to instructions of the manufacturer. Primers 72 777 and W2711X tion if chronic, low-intensity exercise is beneficial or harmful will 72 778 were used for identifying the Flnc and WT alleles have to be addressed in future studies. Furthermore, in this work, (Fig. 1; Supplementary Material, Table S2). we did not examine the cardiac phenotype of our mice, some- thing that should be addressed in the future as a potential way Expression at mRNA level to study the cardiomyopathy element in filaminopathy. RNA was isolated from the soleus muscles of 2-month-old WT and

Taken together, our work has unravelled novel insight into Downloaded from mutant animals using TRIzol reagent (Life Technologies) or the the pathomechanisms underlying MFM-filaminopathy: (i) mu- RNeasy Fibrous Tissue Mini Kit in combination with TissueLyser tant filamin C interferes with mechanical stability and strain re- LT (Qiagen, Hilden, Germany). First-strand cDNA was synthe- sistance of myofibrillar Z-discs; (ii) this reduced stability is the sized using the Superscript First-strand Synthesis System (Life basis of the formation of micro- and macrolesions, which we re- Technologies) or Omniscript RT Kit (Qiagen) with random gard as preclinical disease stages preceding development of the hexamers, according to the manufacturers. Multiplex, semi- characteristic protein aggregates; and (iii) the lesion pathology http://hmg.oxfordjournals.org/ quantitative RT-PCR (25–30 cycles) of filamins and Gapdh as en- (rather than the formation of protein aggregates) may be the dogenous reference was performed using 5X FIREPol Master major contributing factor to muscle weakness experienced by pa- Mix (Solis BioDyne, Tartu, Estonia). For quantification, all reac- tients. Based on these findings, we propose that acute strenuous tions were performed in triplicate, using three different cDNA exercise may significantly enhance progressive myofibrillar dam- samples prepared from different individuals. Bands were quanti- age and thus should be avoided in MFM-filaminopathy patients. fied using ImageJ and normalized to the endogenous reference, before means were calculated and plotted. Materials and Methods Real-time PCR was performed using Applied Biosystems 7500 Real-Time PCR system and Assay on Demand reagents according Generation of the Flnc W2711X knock-in mouse model to the recommendations of the manufacturer (Applied Biosys- at Erlangen Nuernberg University on August 15, 2016 tems). The expression levels obtained were normalized to The knock-in strategy was designed and carried out by genOway that of glyceraldehyde-3-phosphate dehydrogenase (Gapdh). (Lyon, France). The Flnc gene-targeting vector was constructed Assay-on-Demand references for Flnc and Gapdh were Mm from genomic C57Bl/6 mouse strain DNA. The p.W2711X point 00471824_m1 and Mm99999915_g1, respectively. mutation was inserted into Flnc exon 48, while a Neo cassette flanked by Flp recombinase target (FRT) sites was inserted in in- fi tron 46 (Fig. 1B). Infrared western blot analysis and protein quanti cation The linearized targeting vector was transfected into C57Bl/6 Muscle samples were weighed and snap frozen in liquid nitrogen. embryonic stem (ES) cells. G418-resistant clones were isolated, Samples were mechanically disrupted using a TissueLyser LT fi ampli ed and genotyped by PCR and Southern blot analysis. (Qiagen) at 50 Hz and dissolved in 15 µl urea buffer [2 M thiourea, PCR analysis was performed using primers 72 772 and 0069-Neo 7 M urea, 5 m EDTA, 1 m DTT and protease inhibitors (Sigma, (for primer sequences see Supplementary Material, Table S2) P8340) in 100 m Tris pH8.6] per 1 mg of muscle sample by hom- fi fi that speci cally ampli es the targeted locus. The products of a ogenization for 3 min at 50 Hz. Protein concentrations were de- second PCR (using primers 72 797 and 72 798) were sequenced termined using the 2D Quant Kit (GE Healthcare Life Sciences, to validate the presence of the mutation. The targeted locus Freiburg, Germany). SDS sample buffer with a concentration of fi was con rmed by Southern blot analysis using internal and ex- 5× was added to a final concentration of 2×, and samples were in- ′ ′ ternal probes on both 5 and 3 ends. Fourteen clones were iden- cubated for 5 min at 55°C. Proteins were separated by SDS–PAGE fi ti ed as correctly targeted. and transferred onto a polyvinylidenfluorid (PVDF) membrane. Several clones were microinjected into C57Bl/6J-Tyrc-2J/J blas- Membranes were blocked with Odyssey blocking buffer (LI-COR fi tocysts and gave rise to 13 male chimeras with a signi cant ES Biosciences, Bad Homburg, Germany). Primary antibodies diluted cell contribution. These mice were bred to C57BL/6 mice expres- in Tris-buffered saline with 0.05% Tween 20 (TBST) were applied sing Flp recombinase to remove the Neo cassette. Genotyping pri- overnight at 4°C. Subsequently, membranes were washed in mers (72 777, 72 778) were designed to discern between the TBST and incubated with IRDye-680- or -800-conjugated second- different alleles by PCR, and selected animals were further vali- ary antibodies (LI-COR Biosciences). Samples were analysed ′ dated by Southern blot analysis using a 5 external probe. using a LI-COR Odyssey Infrared Imaging System (LI-COR Bios- Mice were housed in isolated ventilated cages (IVC) equipped ciences). Bands were quantified using the Odyssey Infrared Im- fi with spruce granulate embedding and a nest under speci c and aging Software v. 1.2 (LI-COR Biosciences) and normalized to opportunistic pathogen-free (SOPF) conditions at 22 ± 2°C, GAPDH. 50–70% air humidity, 70 air exchanges per hour and a light– dark cycle of 12/12 h with free access to water and food. Litter- Antibodies mates were separated at weaning by sex and housed at maximal five animals per cage. Health monitoring was done as recom- Antibodies used in this study and their dilutions are given in Sup- mended by the Federation of European Laboratory Animal plementary Material, Table S3. A novel rabbit antiserum was 7218 | Human Molecular Genetics, 2015, Vol. 24, No. 25

raised against a peptide representing the C-terminal 16 amino (fluorescein isothiocyanate)-, Alexa fluor 555- or Alexa fluor 647- acids of mouse filamin C that are deleted in mutant mice (Bio- conjugated secondary antibodies (Southern Biotech, Birmingham, , Berlin, Germany). Specificity of affinity purified antibodies AL, USA; Jackson Immunoresearch Laboratories, West Grove, PA, for WT filamin C was confirmed by testing its reactivity on recom- USA; Molecular Probes/Life Technologies, Darmstadt, Germany). binant filamins A and B, and WT and mutant filamin C fragments Slides were mounted with Mowiol mounting medium. Images (Supplementary Material, Fig. S10). were acquired using an LSM710 or LSM780 confocal laser scanning microscope or a Cell Observer SD spinning disc microscope (Carl Clinical phenotyping/grip strength measurement and Zeiss GmbH, Oberkochen, Germany). hanging-wire test Both tests were performed by standard protocols as described Electron microscopy previously (58). A standard EM protocol was used in this study. Briefly, soleus muscle samples from WT and mutant mice (n = 2 per genotype) Acute exercise were fixed in 4% paraformaldehyde, 15% saturated picric acid and 0.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 over- Acute exercise was performed by subjecting 8-month-old WT

night at 4°C. After rinsing in PBS, sections were treated with Downloaded from and mutant male mice to a forced run until exhaustion using a 0.5% OsO4, washed and counterstained with uranyl acetate, de- computerized, electronically controlled level treadmill device hydrated via ethanol series and embedded in Durcupan resin (TSE Treadmill ‘Kombi’ with air puff, TSE Systems, Bad Homburg, (Fluka, Switzerland). Ultrathin sections were prepared (Ultracut Germany). Before the trial, mice were trained at 3 consecutive S; Leica, Germany) and examined with a Zeiss LEO906E or Zeiss days at a maximal speed of 0.1 m/s for 5 min. During the trial, a LEO 910 electron microscope (Carl Zeiss GmbH). starting speed of 0.1 m/s was increased by 0.02 m/s every 2 min http://hmg.oxfordjournals.org/ until mice got exhausted. Subsequently, mice were kept for 24 h in a Phenomaster system (see below), before they were eutha- Statistical analysis nized and muscles were dissected for further studies. Data analyses and statistical evaluations were performed using Excel 2010 (Microsoft), Student’s t-test and Mann–Whitney U Automated phenotyping (Wilcoxon rank-sum) test, using the Mann–Whitney U test calcu- lator at www.socscistatistics.com/tests/mannwhitney. For the Automated phenotyping of mice followed a previously described clinical phenotyping, data were subjected to two factorial (59) and validated (60) approach. For details, see Supplementary ANOVA for repeated measurements, with the inter-individual Material, Methods section. ‘genotype’ and one or more factors depending on the test used Four male WT and four mutant male mice (8 months old) were at Erlangen Nuernberg University on August 15, 2016 (GraphPad). individually observed under a 12 h light–dark cycle before (68 h; Preparation of all figures was done using Corel Draw Graphics three dark phases and two light phases) and directly after (24 h) Suite 12 or X7. acute treadmill exercise. Data were collected automatically in 1 min registration–sample intervals, and pooled in 20 min bins, or in complete dark phases. Data from the second dark phase Supplementary Material were used as pre-exercise reference conditions. A two-way Supplementary Material is available at HMG online. ANOVA for repeated measurements was applied, with ‘genotype’ as inter-individual factor and ‘parameter across time’ (e.g. X + Y ‘ ’ activity, Z-plane rearing activity, vCO2, etc.) as intra-individual Acknowledgements factor. An uncorrected Fisher’s least significance difference test was applied as multiple comparisons test. Prism software vs6 The authors thank Dr Ekaterini Kordeli, Paris, France for her gen- (GraphPad Software, Inc.) was used for all statistics. A critical erous gift of Ankyrin-G antibodies, and Karin Bois and Elke Meyer value for significance of P < 0.05 was used throughout the study. for technical assistance. All data represent means ± SEM. Conflict of Interest statement. None declared. For lesion quantification, muscles from exercised mice were collected directly after the second automated phenotyping. For comparison, muscles from four unexercised 8-month-old male Funding WT and mutant mice were sampled. This work was supported by the Else Kröner-Fresenius-Stiftung (D.O.F. and R.S.); the German Research Foundation (Multilocation Histology, immunochemistry and light microscopy DFG-Research Units FOR1228 and FOR1352, D.O.F., R.A.K. and Muscles were dissected and frozen in liquid nitrogen-cooled iso- R.S.), the Seventh Framework Programme for Research and pentane. Cryostat sections (5 μm thick) were prepared and Technological Development of the EU (MUZIC to D.O.F.), the As- stained with HE, modified Gomori trichrome, NADH (reduced sociation Française contre les Myopathies (F.C.) and the German nicotinamide-adenine dinucleotide) tetrazolium reductase and Research Foundation (Grant No. INST 410/45-1 FUGG) for use of SDH as described (61). the LSM 780 laser confocal microscope (Erlangen). For immunostaining, cryostat sections were air dried for 30 min, fixed with acetone (−20°C) for 10 min, air dried for References 30 min and incubated with blocking medium [3% foetal bovine serum (FBS), 1% goat serum, 0.1% sodium azide in phosphate- 1. Schröder, R. and Schoser, B. (2009) Myofibrillar myopathies: a buffered saline (PBS)] for 1 h at RT. Sections were incubated clinical and myopathological guide. Brain Pathol., 19, 483–492. overnight at 4°C with primary antibodies diluted in 1% blocking 2. Selcen, D. (2011) Myofibrillar myopathies. Neuromuscul. Dis- medium in PBS, and after washing, with the appropriate FITC ord., 21, 161–171. Human Molecular Genetics, 2015, Vol. 24, No. 25 | 7219

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