Myotilin – a prominent marker of myofibrillar remodelling

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/6740186 Myotilin-a prominent marker of myofibrillar remodeling ARTICLE in NEUROMUSCULAR DISORDERS · FEBRUARY 2007 Impact Factor: 2.64 · DOI: 10.1016/j.nmd.2006.09.007 · Source: PubMed CITATIONS READS 19 46 5 AUTHORS, INCLUDING: Lena Carlsson Ji-Guo Yu 15 PUBLICATIONS 835 CITATIONS 23 PUBLICATIONS 481 CITATIONS Umeå University SEE PROFILE Umeå University SEE PROFILE Lars-Eric Thornell Umeå University 316 PUBLICATIONS 9,554 CITATIONS SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately. Available from: Lena Carlsson Retrieved on: 03 February 2016 Neuromuscular Disorders 17 (2007) 61–68 www.elsevier.com/locate/nmd Myotilin – a prominent marker of myofibrillar remodelling Lena Carlsson a, Ji-Guo Yu b, Monica Moza c, Olli Carpén d, Lars-Eric Thornell a,* a c Department of Integrative Medical Biology, Section for Anatomy, Umea University, Sweden b Department of Natural and Environmental Physiology, Mid Sweden University, Sweden Department of Pathology and Neuroscience Program, Biomedicum Helsinki, University of Helsinki and University Central Hospital, Finland d Department of Pathology, University of Turku and Turku University Central Hospital, Finland Received 20 April 2006; received in revised form 25 August 2006; accepted 5 September 2006 Abstract Myofibrillar remodelling with insertion of sarcomeres is a typical feature of biopsies taken from persons suffering of exercise-induced delayed onset muscle soreness. Here we studied the presence of the sarcomeric protein myotilin in eccentric exercise related lesions. Myotilin is a component of sarcomeric Z-discs and it binds several other Z-disc proteins, i.e. a-actinin, filamin C, F-actin and FATZ. Myotilin has previously been shown to be present in nemaline rods and central cores and to be mutated in limb girdle muscular dystrophy 1A (LGMD1A) and in a subset of myofibrillar myopathies, indicating an important role in Z-disc maintenance. Our findings on non-diseased muscle affected by eccentric exercise give new information on how myotilin is associated to myofibrillar components upon remodelling. We show that myotilin was present in increased amount in lesions related to Z-disc streaming and events leading to insertion of new sarcomeres in pre-existing myofibrils and can therefore be used as a marker for myofibrillar remodelling. Interestingly, myotilin is preferentially associated with F-actin rather than with the core Z-disc protein a-actinin during these events. This suggests that myotilin has a key role in the dynamic molecular events mediating myofibrillar assembly in normal and diseased skeletal muscle. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Eccentric exercise; DOMS; Z-disc streaming 1. Introduction Myotilin is a recently recognised protein which in human skeletal muscle is mainly present in the myofibrillar Z-discs [1]. The protein has been named myotilin (myofibrillar protein with titin-like Ig domains) as it has similarities with the Ig domains of titin. More recently, two related proteins, palladin and myopalladin were described, and these three proteins form a novel subfamily of intracellular Ig-domain-containing molecules [2–4]. Myotilin interacts with a-actinin, a key structural protein of the Z-disc [1], and with filamin C (c-filamin) * Corresponding author. Tel.: +46907865142; fax: +46907865480. E-mail address: [email protected] (L.-E. Thornell). 0960-8966/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2006.09.007 [5], F- and G-actin [6,7] and with FATZ (calsarcin, myozenin) [8]. Functional evidence for a role of myotilin in the structure of sarcomere comes from studies in which expression of N- or C-terminal myotilin deletion constructs led to sarcomere disorganization and collapse [5,6,9]. Therefore, myotilin appears to have a key role in the dynamic molecular events mediating myofibril assembly and in the organisation and/or maintenance and integrity of the sarcomeres [6]. Also clinical data indicate an important role for myotilin in sarcomere biology. Missense mutations of myotilin cause limb girdle muscular dystrophy 1A [10], some forms of myofibrillar myopathy (MFM) [11], spheroid body myopathy [12], late onset distal myopathy [13] or a myopathy, which has a clinical/pathological phenotype that appears to be intermediate between that 62 L. Carlsson et al. / Neuromuscular Disorders 17 (2007) 61–68 described in LGMD1A and MFM myotilinopathy [14]. The main morphological features of myotilinopathies consist of myofibrillar lesions, preferentially Z-disc abnormalities. The myofibrillar accumulations characteristic of MFM are intensively labelled with myotilin antibodies [11,14,15]. Furthermore, strong myotilin staining has been observed in other myofibrillar abnormalities such as nemaline rods, central core lesions [16] and spheroid bodies [12]. Interestingly, a transgenic mouse model has been developed which seems to unite the pathology associated with LGMD1A and MFM [17]. Myofibrillar disorganisation with Z-disc streaming, Z-disc smearing and Z-disc disruption are the morphological hallmarks of delayed onset muscle soreness (DOMS) [18]. On basis of animal models thought to mimic DOMS, the cytoskeleton and especially the desmin intermediate filaments have been shown to be early affected and proposed to be a causative factor to myofi- brillar damage [19–21]. In addition, inflammation and necrosis are thought to be involved in the processes of DOMS [18,22]. Based on studies on human subjects, the concept of pathogenesis in DOMS has recently been challenged as, in several studies, desmin appears not degraded and necrosis is not observed [23,24]. On the contrary, affected muscles show signs of an increased synthesis of both desmin and actin [23,24]. In certain areas, increased staining for desmin correlates with a lack of the Z-disc related proteins a-actinin, titin and nebulin, whereas in other areas, an increased number of cross-striations, i.e. supernumerary sarcomeres, stained with a-actinin, titin and nebulin are observed [25]. Based on a combination of results obtained from immunohistochemistry, electron microscopy and immunoelectron microscopy, we have proposed a model where, instead of muscle damage, the myofibrillar alterations reflect an adaptive remodelling of the myofibrils. The process subsequently leads to sarcomerogenesis and Fig. 1. Semi-thin cryosections of longitudinally cut muscle fibres from a biopsy of a normal soleus muscle. (A–C) Low magnification of a section double immunostained with anti-myotilin (A) and the MyHC antibody N2.261, which strongly stains the type II fibres (B) (merged images in (C)). Transversely oriented striations of myotilin are strongly stained in type I fibres and moderately/weakly stained in type II fibres (*). (D–F) High magnification of a muscle fibre double immunostained with anti-myotilin (D) and anti-desmin (E) (merged images in (F)). Strong transverse striations composed of short strands of myotilin are alternating with dots of desmin at the Z-disc level. In between these striations (M-band level) a weak labelling of myotilin is also seen (arrow heads). Scale bars 10 lm. L. Carlsson et al. / Neuromuscular Disorders 17 (2007) 61–68 lengthening of the myofibrils [23–26], providing for the first time a mechanistic explanation to the lack of soreness after additional exercise. As myotilin appears to be an important regulator of the sarcomere, we decided to analyse myotilin and its binding partners a-actinin and F-actin in DOMS. In the present study we show that the distribution of myotilin is dramatically changed in subjects with DOMS. 2. Material and methods 2.1. Subjects and experimental protocol Sixteen healthy men with a mean age of 24.3 years participated in this study. Ten subjects were subjected to a bout of eccentric exercise, whereas the remaining six served as controls. All subjects were asked to refrain from exercise during the experimental period. The exercise group had to run downstairs from the tenth floor to the ground floor, take the elevator back and repeat the procedure fifteen times. The exercise induced severe pain, which reached peak values 2–3 days after the exercise. Open surgical biopsies of the soleus muscle from controls as well as from subjects were taken before exercise 63 and 1 h, 2–3 days or 7–8 days after eccentric training (for details, see [24]). 2.2. Tissue preparation and sectioning The muscle biopsies were stretched on a cork-plate and fixed for 2 h in freshly prepared 2% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS) containing 0.01% glutaraldehyde (GA). After rinsing in PBS, samples were cut into 1-mm cubes and infiltrated in 2.3 M sucrose. Muscle samples were longitudinally oriented and snap frozen on stubs in liquid nitrogen. The specimens were cut into semi-thin (0.2 lm) and ultra-thin (70–100 nm) sections with a Reichert Ultracut microtome, equipped with a FCS cryo attachment (Leica, Nussloch, Germany). Semi-thin sections were collected on chrome-alum-gelatine coated slides and labelled with indirect immunofluorescence for light microscopy. 2.3. Immunofluorescence and light microscopy Semi-thin sections were rehydrated in 0.01 M PBS, immersed in 5% non-immune serum and incubated with primary antibodies for 60 min at 37 °C or overnight at Fig. 2. A semi-thin cryosection of a longitudinally cut soleus muscle biopsy taken 7–8 days after eccentric exercise double immunostained with antimyotilin (A) and anti-desmin (B) (merged images in (C)). (A)–(C) Lesions of varying size are strongly stained with myotilin, whereas desmin shows a fairly normal cross striated staining pattern (in (D)–(F) higher magnification of the upper boxed area in (A)–(C)). Note the difference in staining intensity of myotilin between the different muscle fibres and that a weak staining of myotilin is present at the M-band level (in the middle of the sarcomere) in the top and bottom fibres (*). (G)–(I) Higher magnification of the lower boxed area in (A)–(C) shows that in some areas with strongly stained myotilin plaques desmin is present in transverse as well as in some longitudinal strands (short arrows). Notice that myotilin is unaffected in the sarcomere containing a single longitudinal strand of desmin extending between two transverse lines (long arrow). Scale bars 10 lm in (A)–(C); 4 lm in (D)–(I). 64 L. Carlsson et al. / Neuromuscular Disorders 17 (2007) 61–68 4 °C. Visualisation of bound antibodies was performed with indirect fluorescence using Alexa 488 or fluorescein (FITC) for green fluorescence and Alexa 568 or rhodamine red-X for red fluorescence (Molecular Probes Inc., Eugene, OR, USA, and Jackson Immunoresearch Laboratories, West Grove, PA, USA). Double labelling was performed either sequential or simultaneous with one monoclonal and one polyclonal antibody, followed by secondary antibodies coupled to fluorochromes of different wavelengths. The sections were evaluated in a Nikon eclipse E 800 microscope (Nikon Inc., Melville, NY, USA) and a SPOT RT Color camera (Diagnostic Instruments Inc., Sterling Heights, MI, USA) was used for image acquisition. Digital images were processed using the Adobe Photoshop software (Adobe Systems Inc., Mountain View, CA, USA). Control sections were treated as above, except that the primary antibody was exchanged with non-immune serum. proteins (Fig. 1D–F), whereas sections also stained with phalloidin revealed a Z-disc pattern for myotilin (controls not shown but see unaffected area in Fig. 3). Similarly, double staining with antibodies against myotilin and a-actinin showed a complete colocalisation at the Z-discs (controls not shown but see unaffected area in Figs. 4 and 5). Focal areas with increased staining of myotilin were observed in muscle fibres affected by eccentric exercise (Figs. 2–5). The alterations observed were variable in size, from a single Z-disc affected to large lesions involving several consecutive sarcomeres. Myotilin staining appeared as dots, broad bands, plaques and masses. 2.4. Antibodies and ligands Myotilin was detected with anti-myotilin 151 rabbit polyclonal antibody raised against recombinant N-terminal fragment (residues 1–151) of myotilin [27] and used as a crude antiserum. a-actinin was recognised by the monoclonal antibody EA-53 (Sigma, Copenhagen, Denmark). Myosin heavy chain (MyHC) composition was determined with monoclonal antibodies purchased from Developmental Studies Hybridoma Bank, Iowa City, IA, USA. A4.840 stains slow type I fibres and N2.261 stains fast type IIA fibres strongly and slow type I fibres weakly [28]. Desmin was identified with the monoclonal antibody desmin D33 (Dako, Glostrup, Denmark) or the polyclonal antibody desmin A611 (Dako, Carpinteria, CA, USA). Direct-conjugated rhodamine phalloidin (red) (Molecular Probes Inc., Eugene, OR, USA) or Alexa fluor 488 phalloidin (green), which binds to actin filaments served as a marker for myofibrils. 3. Results The antibody against myotilin stained all myofibres/ myofibrils from controls and unaffected fibres/fibre areas from exercised subjects in regular transverse striations (Fig. 1). The staining, which was seen as short transverse strands, was strong in type I fibres, whereas type II fibres were moderately stained (Fig. 1A–C). The fibre types were determined with different MyHC antibodies. Occasionally, a weak staining of myotilin was also present at the M-band level (Fig. 1D and F). The precise myofibrillar localisation of myotilin was revealed by double immunostainings with antibodies against myotilin and other well-known sarcomeric components. Sections double stained with anti-desmin and myotilin showed an alternating dotted pattern for these Fig. 3. A semi-thin cryosection of a longitudinally cut soleus muscle biopsy taken 7–8 days after eccentric exercise double immunostained with anti-myotilin (A) and phalloidin (B) (merged images in (C)). Insets show higher magnification of *-marked areas. Strong staining of myotilin and phalloidin are largely colocalised. Note, however, that their distribution differs to some extent. The staining of phalloidin is seen as strands extending from one Z-disc to another Z-disc, whereas the staining for myotilin is strongest at the Z-discs and more diffusely spread over the sarcomere. Note also that an unequal number of sarcomeres are present in affected areas (arrowheads). Scale bar 10 lm. L. Carlsson et al. / Neuromuscular Disorders 17 (2007) 61–68 65 Fig. 4. A semi-thin cryosection of a longitudinally cut muscle fibre from a soleus muscle biopsy taken 2 days after eccentric exercise double immunostained with anti-myotilin (A) and anti-a-actinin (B) (merged images in (C)). A large part of the myofibrils show strong staining for myotilin, whereas the staining for a-actinin is decreased. Scale bar 10 lm. The relationship between the staining pattern of myotilin and desmin, a-actinin, myosin and actin was studied by double immunostainings. The increased staining for myotilin did not correlate with an increased labelling for desmin (Fig. 2A–I). For example, in an area extending over more than 20 sarcomeres with strong staining for myotilin, an equivalent increase in the staining for desmin was not observed, nor were longitudinal strands of desmin present (Fig. 2A–I). However, upon detailed examination, desmin staining was present at the borders of the individual broad Z-disc plaques strongly stained for myotilin (Fig. 2G–I). In addition, thin longitudinal strands of desmin staining were observed in the vicinity of the plaques (Fig. 2D–I). In other areas, the longitudinal links of desmin were lacking colocalisation of myotilin (Fig. 2G–I). The distribution of F-actin and myotilin was compared by double staining with myotilin and the F-actin probe phalloidin. Enriched phalloidin staining was typ- ical for all areas with strong myotilin staining (Fig. 3A– C). The two staining patterns largely colocalised. In general, phalloidin staining was seen as strands extending from one Z-disc to another, whereas myotilin staining was strongest at the Z-discs and more diffusely spread over the sarcomere. Staining for a-actinin was in general weaker or partially lacking in areas of strong staining for myotilin (Figs. 4 and 5). In some broadened sarcomeres stained for myotilin, a weak transverse line stained for a-actinin was observed dividing the sarcomere into two parts, which differ with respect to myotilin staining. One part lacked myotilin and the other part was strongly stained (Fig. 5C–E). 4. Discussion Our results confirm that myotilin colocalises with a-actinin and actin within the myofibrillar Z-discs of 66 L. Carlsson et al. / Neuromuscular Disorders 17 (2007) 61–68 Fig. 5. A semi-thin cryosection (A–C) of a longitudinally cut soleus muscle biopsy taken 7–8 days after eccentric exercise double immunostained with anti-myotilin (A) and anti-a-actinin (B) (merged images in (C)). A broadened sarcomere is strongly stained for myotilin and shows a weak line of staining for a-actinin in the middle (arrowheads in (B)). In the merged image (C) the a-actinin stained line can be seen to be enclosed by high amount of myotilin staining on one side and no staining on the other. Scale bars 5 lm. normal muscle fibres [1,6]. Furthermore, we show that the staining of myotilin is more abundant in type I than in type II fibres. We suggest that the difference in degree of staining might be related to the width of the Z-discs. It has been shown that type I fibres have broader Z-discs than type II fibres [29]. The width of the Z-discs is generally explained to reflect differences in the number of crossbridge repeats linking the F-actin filaments of opposing sarcomeres together [30]. The crossbridges are thought to be formed by a pair of a-actinin molecules. How myotilin is exactly organized in the Z-disc is not known, but myotilin binds to a-actinin [1], filamin C [5], F-actin [6] and FATZ [8]. Previously a fibre type dependent myotilin staining has not been reported. Our sectioning and staining methodology gives improved preservation of the tissue and allows thinner sections to be cut, which might make epitopes on the proteins more accessible for staining. However, we observed fibre type related differences in staining also in ordinary cryostat sections. The use of very thin sections further means less background staining and absent or less overlapping of stained structures, which further enhance resolution [23]. Another novel observation in normal muscle fibres was the presence of some staining of myotilin at the M-band level in some specimens. The staining was always much lower in intensity than that of the Z-discs. We did not observe staining for myotilin at the sarcolemma, which has been reported by Salmikangas [1] and Schröder [16]. Our observation is in agreement with that of Selcen [15], who also failed to detect evident staining of myotilin at the sarcolemma in muscle biopsies of patients with myofibrillar myopathies. In myofibrillar alterations induced by eccentric exercise, myotilin staining is markedly increased. The staining can be seen in one single broadened Z-disc up to large lesions involving several consecutive sarcomeres. The staining coexists with areas showing increased amount of phalloidin staining. Our recently published papers showed that the increase of phalloidin staining occurs in areas with a temporary lack of a-actinin, as well as of two other Z-disc related proteins, titin and nebulin [23,25]. This observation strongly suggests that myotilin is more associated to F-actin than to a-actinin in myofibrillar alterations related to eccentric exercise and DOMS. This is in accordance with that maximal binding between myotilin and F-actin is obtained at 1:1 molar ratio as evidenced by co-sedimentation assays [6]. In addition to the actin binding ability, the immunoglobulin domains of myotilin are also bundling domains, which makes myotilin able to dimerise and might thereby contribute to a higher stability of the Factin filaments within phalloidin-stained areas. Moreover, using pull-down assays myotilin has been shown to bind also to G-actin [7]. Myotilin could therefore be an important player during initial steps of the remodelling process by recruiting G-actin to the a-actinin devoid Z-discs. Our present study shows that myotilin so far is the most prominent marker for myofibrillar remodelling caused by eccentric exercise. The antibody against myotilin detected minimal changes as broadenings of the Zdiscs in individual sarcomeres. Additional sequences of events can now be postulated in agreement with our previously proposed scheme for sarcomerogenesis [25]. New sarcomeres can be formed in two ways, either by a split of an existing Z-disc or by reintegration of a-actinin and related Z-disc proteins (titin and nebulin) in a single broadened sarcomere (Fig. 6). Z-disc streaming and other myofibrillar abnormalities are common features of many myopathies [31]. Our preliminary results indicate that myotilin is an equally good marker for such alterations. This is in accordance with the observation that staining for myotilin was the best marker for diagnosis of myofibrillar myopathies [15], in which 90% of the fibres were stained excessively for myotilin, whereas disturbed staining for desmin was only seen in 75 % of the fibres. Our observations on myofibrillar remodelling rule out a direct relationship between myotilin and the desmin cytoskeleton, as increased staining of these two proteins was completely independent of each other. L. Carlsson et al. / Neuromuscular Disorders 17 (2007) 61–68 67 Fig. 6. Schematic models of myofibrillar remodelling induced by eccentric exercise. A new sarcomere can be formed either by a split of a single Z-disc (A) or by an alteration of a single sarcomere (B). In the model the major constituents of a myofibril are shown: Myosin (black), actin (dark blue), titin (grey), nebulin (light blue), a-actinin (dark red), desmin (green) and myotilin (red). A1: two normal sarcomeres are shown; A2: a-actinin is lost, and F-actin and myotilin are increased in the middle Z-disc. A3: The Z-disc is broadened: F-actin and myotilin are increased and a-actinin is present at the edges of the broadened Z disc. A4: A new sarcomere is formed with insertion of myosin, titin and nebulin. Note that desmin in the areas of remodelling is increased and also present as longitudinal strands. B1: A normal sarcomere is shown. B2: The sarcomere is broadened. F-actin, myotilin and desmin are increased, and a-actinin, myosin, titin and nebulin are lost. B3: a-Actinin is reintegrated in the middle of the broadened sarcomere forming a new Z-disc. Subsequent addition of the other myofibrillar proteins is not coordinated in the two sarcomeres. B4: An additional sarcomere has been added to the myofibril. Acknowledgements We wish to thank Mrs. M. Enerstedt for excellent technical assistance. This work was supported by grants from the Swedish National Centre for Research in Sports (98/04, 108/05), the Swedish Research Council (12X-3934) and the Medical Faculty of Umeå University to L-ET; and by the Sigrid Juselius Foundation, the Academy of Finland and the Finnish Heart Research Foundation to O.C. References [1] Salmikangas P, Mykkänen OM, Grönholm M, et al. Myotilin, a novel sarcomeric protein with two Ig-like domains, is encoded by a candidate gene for limb-girdle muscular dystrophy. Hum Mol Genet 1999;8:1329–36. [2] Bang ML, Mudry RE, McElhinny AS, et al. Myopalladin, a novel 145-kDa sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies. J Cell Biol 2001;153:413–27. [3] Mykkänen OM, Grönholm M, Ronty M, et al. Characterization of human palladin, a microfilament-associated protein. Mol Biol Cell 2001;12:3060–73. [4] Parast MM, Otey CA. Characterization of palladin, a novel protein localized to stress fibers and cell adhesions. 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