Introduction
Hereditary myopathies are a clinically and genetically heterogeneous group of disorders with a variable age of onset, from congenital to late adulthood. The myopathies have been classified into subgroups based on the clinical distribution of muscle weakness (e.g. proximal vs. distal) and the inheritance pattern (e.g. autosomal dominant vs. recessive), before the genetic etiology was revealed [
43]. Today, mutations in dozens of different genes are known to cause a myopathy [
27], with Duchenne muscular dystrophy (DMD) due to mutations in dystrophin being the most frequent. A large subgroup encompasses the limb-girdle muscular dystrophies (LGMDs) caused by mutations in at least 30 different genes with autosomal dominant (LGMD1; 9 genes/loci) or autosomal recessive (LGMD2; 24 genes) inheritance [
57]. One of the most frequent subtypes of LGMD is LGMD2A caused by homozygous or compound heterozygous mutations in the gene encoding the proteolytic enzyme calpain-3 (CAPN3), a known interactor of the giant sarcomere protein titin [
16,
48]. Myofibrillar myopathies (MFMs), another group of hereditary muscle disorders, are characterized by histological features including focal disintegration of myofibrils and protein aggregation in myofibers. Known MFM disease genes encode proteins associated with the sarcomeric Z-disc, such as myotilin, desmin, and filamin-C [
29]. For many LGMDs and MFMs, the molecular mechanisms underlying the respective disorder remain largely unresolved and specific or ameliorating therapies are not available.
In our previous approaches to this topic, we focused mainly on mechanisms of protein quality control, which we found to be pathologically altered in MFMs [
30]. We deciphered distinct mutation-specific disease mechanisms in the human myopathies, such as protein misfolding and aggregation, toxic gain of function, and haploinsufficiency [
2,
21]. Recently, we demonstrated that the small heat shock proteins (sHSPs), HSP27 (HSPB1) and αB-crystallin (HSPB5), which in healthy muscle cells localize to the Z-disc or cytosol, were translocated to the titin springs of the sarcomeric I-bands in LGMD2A myocytes [
31]. Titin is established as being responsible for the elasticity and “passive” tension (PT) of the myocyte [
39], but is also evolving as a protein critical for the active mechanical properties of the sarcomere [
26,
36,
52]. The binding of HSPs to titin suggested a role for these molecular chaperones in the pathomechanism of myopathy subtypes, which are presenting with reduced contractile force generation and increased muscle stiffness.
HSPs are important components of protein quality control, as they affect protein folding and promote degradation, e.g. via the ubiquitin-proteasome system (UPS) or autophagy pathways [
7,
56]. Members of the family of sHSPs assist in the folding and maintenance of the cytoplasmic proteome and are considered holdases rather than foldases [
5,
42]. Interestingly, overexpression of sHSPs significantly reduces aberrant protein aggregation in cell and animal models of MFM [
10,
53,
54,
59]. Moreover, chemical chaperones can impede pathological protein aggregation and improve muscle function [
60]. We found that sHSPs stabilize folded immunoglobulin-like (Ig) domains of titin from the elastic I-band segment [
9]. If these Ig modules unfold in response to sarcomere stretching [
1,
52], the sHSPs may help protect them from aggregation [
31]. The ATP-dependent chaperone HSP90 is known to assist in the assembly of the myosin filaments [
55] and alter myosin motor function [
47]. HSP90 also binds, if methylated by the methyltransferase Smyd2, to the N2A element of I-band titin (near the calpain-3-binding site) and exerts a protective effect on Z-disc/I-band structure [
15,
58]. HSP90 is among the chaperones discussed as potential ameliorators of dystrophic muscle disease [
8]. However, its relevance in LGMDs and MFMs has not been studied.
Considering the potential for chaperones to improve myocyte function in muscle disease, we initiated this study with the aim to better understand the relationship between titin and HSPs in human hereditary myopathies. We set out to determine which chaperones associate with titin in muscle biopsies from different myopathies, including LGMD2A and MFM-filaminopathy. We found that, of all HSPs studied, only HSP27, αB-crystallin and HSP90 were translocated from the cytosol or sarcomeric Z-disc in healthy human muscles to the titin springs in myopathy. We mapped the interaction sites by immunoelectron microscopy and measured the impact of endogenous HSP-binding to the sarcomeres on myofiber PT, in controls and myopathy patients. We also tested whether exogenous HSPs added to permeabilized human myofibers affect PT. By examining biopsy material from control subjects, 17 patients with different myopathies, and muscle from animal models of hereditary myopathies, we found that massive HSP-binding to titin is a common feature in dystrophic and MFM muscle disorders. We conclude that the translocation of HSPs to titin, while protecting the protein in the sarcomeres, could also impair titin-based myofiber elasticity, presumably contributing to increased muscle stiffness. These alterations represent a previously unrecognized pathophenomenon in hereditary myopathies.
Methods
Human muscle biopsies
We studied M. vastus lateralis and gastrocnemius biopsies from three healthy (CTRL) subjects with normal histopathological features and 17 diseased human subjects with various muscle disorders (see Table
1). The following hereditary myopathies were included (mutated gene and specific mutation(s) listed in parentheses): Among the group of muscular dystrophies, LGMD2A (
CAPN3; p.Thr184Argfs & Trp130 > Cys, p.Thr184Argfs & p.Arg315Arg, p.Thr184Argfs & p.Gly329Arg) and Duchenne muscular dystrophy (
DMD; Del. exon 2–18, Del. exon 3–11); among the group of myofibrillar myopathies, filaminopathy (
FLNC; p.Val930_Thr/933del (2×)), desminopathy (
DES; p.Arg350Pro), myotilinopathy (
MYOT; p.Ser60Phe, p.Lys36Glu), and a titinopathy, hereditary myopathy with early respiratory failure (HMERF) (
TTN; p.Cys30071Arg (2×)); hereditary inclusion body myopathy (inclusion body myopathy with Paget disease and frontotemporal dementia, IBMPFD) caused by valosin-containing protein mutation (
VCP; p.Arg93Cys, p.Arg155His). At least two biopsy samples per disorder (in filaminopathy from two siblings) were analyzed, with the exception of desminopathy, from which only a single biopsy sample was available. Additionally, we included biopsies from three patients with acquired sporadic inclusion body myositis.
Table 1
Overview of human and mouse muscle samples studied and intracellular localization of major chaperones
Human muscles |
–
| (3×) Healthy CTRL | 1953/72/78/M | Z-disc | Z-disc/Cytosol | Cytosol |
CAPN3
| LGMD2Aa/Calpainopathy | | I-band | I-band | I-band |
| 1) p.Thr184Argfs & Trp130 > Cys | 1967/M | | | |
| 2) p.Thr184Argfs & p.Arg315Arg | 1957/M | | | |
3) p.Thr184Argfs & p.Gly329Arg | 1968/F | | | |
DMD
| Duchenne muscular dystrophy | | I-band | I-band | I-band |
1) Del. exon 2–18 | 1998/M | | | |
2) Del. exon 3–11 | 2014/M | | | |
FLNC
| Filaminopathy (2×) p.Val930_Thr/933del | 1960/M 1960/F | I-band | I-band | I-band |
TTN
| Titinopathy/HMERFb
(2×) p.Cys30071Arg | Unknown/Unknown | I-band | I-band | I-band |
MYOT
| Myotilinopathy | | I-band | I-band | I-band |
1) p.Ser60Phe | 1928/M | | | |
2) p.Lys36Glu | 1940/F | | | |
VCP
| IBMPFDc
(Valosin-containing protein) | | I-band | I-band | I-band |
1) p.Arg93Cys | 1971/M | | | |
2) p.Arg155His | Unknown/M | | | |
DES
| Desminopathy p.Arg350Pro | 1976/M | Cytosol | Cytosol | I-band/ Cytosol |
–
| (3×) Sporadic inclusion body myositis (sIBM) | 1955/1998/2014/M | Z-disc | Z-disc/Cytosol | Cytosol |
Mouse muscles |
–
| Litter-matched WT CONTROLS | 2016/M | Z-disc | Z-disc/Cytosol | Cytosol |
FLNC
| MFM filaminopathy p.W2711X | 2016/M | I-band | I-band | I-band |
DMD
| mdx C57BL/10ScSn | 2016/M | I-band | I-band | I-band |
Ethics, consent and permissions
Patients consented to participate in this study, which conforms to the principles outlined in the declaration of Helsinki and was approved by the ethics committee at Ruhr University Bochum (entries 3447–09 and 3483–09).
Mouse models of hereditary myopathies
Skeletal muscle samples were obtained from two published mouse models of hereditary myopathies, the MFM-filaminopathy mouse (
FLNC, p.W2711X; [
12]) and the mdx mouse (C57BL/10ScSn), the latter of which was a kind gift from Dr. Jens Schmidt (Göttingen, Germany). Littermate wildtype (WT) mouse muscles served as controls. Four (mdx model) and six (FLNC model) animals per group, respectively, were studied.
Passive tension measurements
Force measurements were done according to published protocols [
51]) on isolated skinned muscle fibers from CTRL (2 subjects, 20 fibers), LGMD2A (2 subjects, 12 fibers) and MFM-filaminopathy (2 patients, 15 fibers) biopsies. Deep-frozen biopsy tissue was defrosted and skinned overnight in ice-cold low ionic-strength buffer (75 mM KCl, 10 mM Tris, 2 mM MgCl
2, 2 mM EGTA, and 40 μg/ml protease inhibitor leupeptin, pH 7.2) supplemented with 0.5% Triton X-100. Under a binocular (Leica, Mannheim, Germany), single muscle fibers were dissected and suspended between two mini forceps attached to a piezomotor and a force transducer (Scientific Instruments, Heidelberg, Germany). Force measurements were carried out in relaxing buffer (8 mM ATP, 20 mM imidazole, 4 mM EGTA, 12 mM magnesium propionate, 97 mM potassium propionate, pH 7.2) at room temperature. Stretching of fibers was done stepwise from slack length in 6 quick steps. Following each step the fiber was held at a constant length for 60 s to allow for stress relaxation. After the last step-hold, the fiber was released back to slack length. Sarcomere length (SL) was measured by laser diffraction. Passive force-SL recordings were also performed in the presence of recombinant sHSP. Briefly, the 6-ramp step stretch protocol was first carried out twice in the absence of sHSP and then repeated twice in the presence of 100 μM αB-crystallin or HSP27 (10 μM sHSP showed no effect on PT). For data analysis we considered only the peak force levels at the end of each step, which represents a mixture of elastic and viscous forces. Force was related to the cross-sectional area inferred from the diameter of the specimens (at slack length), to obtain PT. Mean data points and SEM were calculated and fit with a simple polynomial. Some samples were chemically fixed (see below) immediately after mechanical measurements, usually at a stretched length, and studied for endogenous vs. exogenous sHSP localization by indirect immunofluorescence.
Immunofluorescence microscopy
Muscle biopsy samples were fixed in 4% paraformaldehyde (PFA), 15% saturated picric acid in 100 mM phosphate buffered saline (PBS) overnight at 4 °C, dehydrated via ascending ethanol series and embedded in paraffin. Thin sections (5–7 μm) were cut with an RM 2235 Leica microtome (Mannheim, Germany). Sections were rehydrated, blocked in peroxidase blocking buffer, and a citrate-EGTA antigen recovery protocol was performed. Slides were rinsed with PBS and then blocked with 5% bovine serum albumin including 0.5% Triton X-100 for 60 min. Subsequently, sections were incubated with primary antibodies overnight at 4 °C, using one of the following antibodies against (all dilutions in PBS): HSP20 (ab125125, Abcam; 1:1000), HSP22 (ab151552, Abcam; 1:150), HSP27 (Clone 2B4, LSBio; 1:100), HSP27 (SMC 1615D12, StressMarq; 1:250), HSP27 (SR B800, MBL; 1:200), αB-crystallin (SMC 1653A10, StressMarq; 1:250), αB-crystallin (SR 223F, MBL; 1:400), HSP40 (Abcam, ab78437; 1:100), HSP70 (Novocastra/Leica; 1:20) HSP70 (HSPA2, Sigma HPA000798; 1:200), HSPA5 (78 kD glucose-regulated protein, Sigma, HPA038845; 1:500), Hsc70 (Heat shock cognate 71 kDa protein, Abcam, ab2788; 1:300), HSP90 (C45G5, Cell Signaling; 1:400), HSP90α (custom-made by PINEDA Berlin, 1:500), titin 9D10 PEVK (Hybridoma Bank, Iowa City, USA; 1:200), and titin custom-made against PEVK C-terminal segment (Eurogentec, polyclonal, affinity-purified; 1:500). Secondary antibodies were Cy3- or FITC-conjugated IgG (Rockland; 1:400), which were incubated overnight at 4 °C. For endogenous vs. exogenous HSP localization in mechanically stretched myofibers, we used a 6xHIS-conjugated IgG (Abcam ab9136; 1:400) antibody incubated overnight at 4 °C. Stained samples were embedded in Mowiol supplemented with N-propyl-gallate for bleaching protection and analyzed by confocal laser scanning microscopy (Nikon, Eclipse Ti), using a 63× oil Plan-Apochromat objective.
Immunoelectron microscopy
4% PFA fixed biopsy samples were cut into longitudinal 50-μm-thick sections using a VT 1000S Leica vibratome (Mannheim, Germany) and rinsed twice in PBS. Then, the muscle samples were blocked in 20% normal goat serum (NGS) for 1 h and incubated with primary antibodies in PBS supplemented with 5% NGS overnight at 4 °C. In addition to some of the primary antibodies used for indirect immunofluorescence (see above section), we also used anti-titin antibodies T12 (1:100; [
22]) and N2A (custom-made by Eurogentec, Belgium, 1:500; [
37]). The sections were then triple washed with PBS and incubated with 1.4 nm gold-coupled secondary antibodies (Nanoprobes, Stony Brook, NY, USA) overnight at 4 °C. After extensive washing, all sections were postfixed in 1% glutaraldehyde for 10 min and after rinsing, sections were reacted with HQ Silver kit (Nanoprobes). After treatment with OsO
4, samples were counterstained with uranyl acetate in 70% ETOH, dehydrated and embedded in Durcupan resin (Fluka, Switzerland). Resin blocks were made and ultrathin sections prepared with a Leica Ultracut S (Mannheim, Germany) and adsorbed onto glow-discharged Formvar-carbon-coated copper grids. Microscopy was performed on a Zeiss LEO 910 electron microscope and images were taken with a TRS sharpeye CCD Camera (Troendle, Moorenwies, Germany). For some images, we measured the nearest distance across the sarcomeric Z-disc between the mean epitope positions of αB-crystallin, HSP27, HSP90, titin T12, titin N2A, and titin PEVK antibodies using ImageJ, as described previously [
38]. The distance between epitopes was plotted against SL, and data points for each antibody type were fit by two-order regression. At least 10 different cells and 30 sarcomeres per experimental condition were included in the analysis.
SDS-PAGE and immunoblotting
Deep-frozen biopsy tissue was homogenized in modified Laemmli buffer, stored on ice for 10 min and subsequently boiled for 10 min at 97 °C. The protein concentration was determined by spectroscopy using Neuhoff standard protocols, SDS–PAGE was carried out using the Laemmli buffer system in slab gels containing 12.5% polyacrylamide. For immunoblot analysis the proteins were transferred onto nitro-cellulose membranes by semidry electroblotting. The blots were transiently stained with Ponceau S to monitor transfer efficiency, then washed with Tris-buffered saline, and incubated for 2 h with a primary antibody. Chromogenic blotting with alkaline phosphatase conjugated secondary antibodies with nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate was used to visualize chaperone expression on Western blots, using the following antibodies: anti-HSP27 (SR B800, MBL; concentration, 1:100), anti-αB-crystallin (SR 223F, MBL; 1:200), anti-HSP90α (PINEDA Berlin; 1:1000), and anti-β-actin (AC-15 Sigma; 1:100). For measurements of titin:myosin heavy chain (MHC) ratio, homogenized skeletal muscle biopsy samples were analyzed by 2.5% SDS–PAGE, as described [
46]. For titin phosphorylation analysis, 1.8% SDS–PAGE was performed as described [
23]. Global titin phosphorylation was determined by anti-phosphoserine/−threonine antibodies (catalogue No. PP2551 (ECM Biosciences); Biotrend Chemicals, Cologne, Germany). To detect site-specific titin phosphorylation, we used custom-made affinity-purified phospho-serine specific antibodies against pS11878 and pS12022 in the PEVK domain of human titin (custom-made by Eurogentec, Belgium; 1:500). As secondary antibody, we used horseradish peroxidase-conjugated IgG (Acris Antibodies, Herford, Germany). For signal amplification we used the enhanced chemiluminescence Western blot detection kit (GE Healthcare). Staining was visualized using the LAS-4000 Image Reader (Fuji Science Imaging Systems) and densitometry was performed using the manufacturer’s MultiGauge analysis software or ImageQuantTL (GE Healthcare). The signal on the Coomassie-stained PVDF membrane served as a means to detect total protein load (in lieu of a reference protein in the titin size range), and immunoblot signals were normalized to the corresponding PVDF signals. Finally, mean signals obtained for diseased muscle tissues were indexed to signals measured in control muscles.
Statistical analysis
Mean values of PT at a given sarcomere length were compared using two-tailed Student’s t-test. Normal distribution of data was a requirement, as was the passing of the equal variance test. Mean densitometric values obtained from stained gels/Western blots were indexed to the respective mean values of human CTRL muscles and compared using Bonferroni adjusted t-test following ANOVA.
Discussion
Increased muscle stiffness is frequently seen in patients with acquired or inherited myopathies, next to muscle weakness and atrophy as the main symptoms in these disorders. Pathological increases in passive muscle stiffness were observed in DMD patients [
14,
32,
33] and greatly elevated myofiber PT was reported for patients with spasticity caused by impairment of the central nervous system [
20,
49] or patients with facio-scapulohumeral muscular dystrophy [
35]. However, the mechanisms behind these alterations in passive mechanical properties have remained largely unknown. Here, we measured the PT of isolated myofibers from two groups of hereditary myopathies, LGMD2A and MFM-filaminopathy patients, and detected approximately 25% higher levels in either group compared to healthy human myofibers. We excluded titin-isoform transitions and titin phosphorylation changes as causes of this increase. Instead, we found that the PT rise in myopathic muscles is due, at least in part, to intracellular translocation of chaperones to the sarcomeric titin springs, which were devoid of chaperones in healthy muscles. The HSPs that were translocated to I-band titin in myopathy included the two sHSPs HSP27 and αB-crystallin and the ATP-dependent chaperone HSP90. Importantly, we demonstrated that binding of these chaperones to elastic titin is common to hereditary skeletal muscle disorders, but not in acquired human sIBM. We found that the I-band titin-binding pattern of chaperones also appears in mouse models of dystrophic and MFM myopathies, but not in normal WT mouse muscles.
HSP27 and αB-crystallin were shown earlier to translocate to the sarcomeric Z-disc/I-band region of skeletal myofibers under stress conditions. The diverse stressors included intense exercise [
50], myofibril stretching [
31] or disease conditions, such as neurogenic atrophy and central core disease [
18]. The cause of this translocation is incompletely understood. Potential triggers could be intracellular oxidative stress and acidosis [
3,
34], possibly resulting from massive mitochondrial alterations, such as those observed by us in all myopathy samples. Acidic conditions directly affect the sHSPs by promoting the formation and accumulation of large oligomers, thereby increasing chaperone activity [
11,
17]. While reduced pH boosts the aggregation of many sHSP substrates, it also increases the sHSP-mediated protection from aggregation [
3,
6]. Interestingly, acidosis raises the passive stiffness of skeletal muscles [
44]. Although intracellular pH and oxidative stress were not measured in our biopsy samples, it is reasonable to speculate that exercise increases these parameters more in myopathic than in healthy muscles, which may then cause higher chaperone activity (possibly towards elastic titin) in the diseased cells.
A likely trigger for the translocation is the increased expression of sHSPs, which is typical for skeletal myocytes exposed to different stresses [
30,
50]. High levels of sHSPs are beneficial, as they protect cells from oxidative stress, acidosis, energy depletion, and other unfavorable conditions [
45]. In the hereditary dystrophic and MFM muscles studied by us, the expression levels of HSP27 and αB-crystallin were much higher than in normal control muscles. Consequently, the sarcomeres could represent a “sink” for excessive amounts of sHSPs expressed in the diseased myocytes. A proportion of this surplus of chaperone protein may be trapped by “sticky” hydrophobic regions of the sarcomeric I-bands.
We recently showed that sarcomere stretching promotes the unfolding of titin Ig domains in the I-band [
52], which results in the exposure of previously concealed hydrophobic titin regions, to which the sHSPs preferentially bind [
31]. The phosphorylation state of the sHSPs, known to be relevant for their interaction with some substrates, does not seem to alter the interaction with titin domains [
19,
31]. In the LGMD2A and MFM-filaminopathy samples studied in the present work, we detected HSP27 spread out along the proximal/middle tandem-Ig segment of I-band titin. This segment contains many relatively weak domains that unfold under physiological stretch forces [
52]. AlphaB-crystallin was found to be restricted to a narrower region near/at the N2A element of titin, which also comprises Ig domains. In contrast, the sHSP-binding spared titin’s PEVK domain, a permanently unfolded (disordered) region, and the distal tandem-Ig region, which contains more stable Ig domains that rarely unfold under physiological stretch forces [
52]. Assuming that sHSP-binding to the sarcomeric I-bands may be an indirect measure of the unfolded state of the titin Ig domains, our findings implicate increased unfolding of proximal/middle Ig domains in hereditary myopathy patients, possibly due to higher I-band strain than in normal myofibers. In summary, the increased association of sHSPs with the sarcomeric I-bands in myofibers of hereditary myopathy patients likely reflects increased interaction with unfolded titin Ig domains.
Conceptually, unfolding of the proximal/middle Ig domains of I-band titin raises their risk for irreversible aggregation, whereas sHSP-binding lowers this risk and protects the sarcomere [
31]. Small HSPs are known to capture up to an equal weight of (partially) denatured protein before it aggregates [
5]. Thus, sHSPs keep the substrate accessible to other members of the protein quality-control network, notably ATP-dependent chaperones, which are required for subsequent substrate refolding [
42]. If refolding to the native state is not possible, the substrate is likely to be degraded. Hence, the binding of sHSPs to titin Ig domains could maintain the domains in a state that allows their efficient refolding. However, the binding could also be indicative of increased titin protein degradation and turnover in myopathic fibers. Either way, the sHSPs will have an important role in avoiding titin loss-of-function and preserving sarcomeric and muscle functions.
Interestingly, among the proteins overrepresented in protein aggregates of three MFM types (myotilinopathy, desminopathy and filaminopathy), there were many sarcomeric and other cytoskeletal proteins, especially Z-disc proteins, as well as various heat shock proteins (including HSP27 and αB-crystallin), but not titin [
28,
40,
41]. In light of our results, it appears that misfolded/aberrant and potentially toxic titin is not “disposed” in aggregates, like many other cytoskeletal proteins in MFM. Instead, the sHSPs may help maintain titin in the sarcomere in a (partially) functional state, in order to preserve its role as the backbone of the sarcomere in the diseased myocyte. A deviation from this pattern of titin protection by sHSPs was observed only in the single desminopathy patient studied by us. In this biopsy sample, both HSP27 and αB-crystallin were mainly found in aggregates, the defining pathological features of this MFM. Currently we do not know why this patient muscle lacked the I-band binding pattern of sHSPs characteristic of the other myopathy types. Additional desminopathy patient samples should be studied to address this issue.
The presumed protective effect of the sHSPs on titin in most dystrophic and MFM disorders comes at the price of modestly increased passive muscle stiffness. This was suggested by the higher myofiber PT following binding of exogenous sHSPs to elastic titin in controls, but not LGMD2A fibers (which had higher PT than controls before the incubation with sHSPs). Moreover, the sHSPs can interact with and stabilize the folded Ig domains of the titin spring, which would further increase titin-based PT [
9]. Because titin-dependent PT modulates the active contractile properties of skeletal myofibers [
26,
36,
52], the increased PT observed in human myopathy presumably affects, to some degree, the developed tension of patient muscles. We conclude that there is a trade-off between beneficial (protection of unfolded protein) and detrimental effects (mechanical impairment) of sHSP-binding to I-band titin on sarcomere function, with consequences for overall muscle performance in myopathy.
Apart from the sHSPs, we studied a set of other chaperones for their intracellular localization in myopathic versus control muscles. However, the only chaperone that also showed a differential binding pattern was HSP90. This ATP-dependent HSP was mainly in the cytosol in controls and translocated to I-band titin in all hereditary dystrophic and MFM human samples, as well as in the DMD and MFM-filaminopathy mouse models, but not in acquired sIBM. Up to half of the cytoplasmic pool of HSP90 was associated with elastic titin (at the position of the N2A element) in human LGMD2A and MFM-filaminopathy myofibers. This binding pattern might indicate increased proteasomal degradation of I-band titin fragments. Importantly, HSP90 did not show significantly altered expression levels in the diseased muscles. Thus, a negative effect of the massive translocation of HSP90 to the titin springs could be the lack of this chaperone in myocyte compartments where it is usually required to perform its many chaperoning tasks. These findings support a concept [
8] whereby the induction of HSP90 in myopathic muscles could be useful to ameliorate some pathological features in the patients.
A mechanistic link between HSP90 and sarcomeric proteins is evident from previous work. HSP90 is known as an essential modulator of myofibril organization and thick filament assembly [
4,
13,
24]. Although much of the evidence is related to its association with myosin, HSP90 also binds to the N2A element of titin if methylated by a co-chaperone, the methyltransferase Smyd2 [
15]. The Smyd2–methyl-HSP90 complex stabilizes the N2A element and helps maintain the sarcomeric Z-disc/I-band structure, which benefits muscle contraction [
15,
58]. Because methylation of HSP90 by Smyd2 could be a trigger for the translocation of HSP90 to elastic titin in hereditary myopathies, we included Smyd2 in our immunoelectron/immunofluorescence microscopical analysis of WT and mdx mouse muscles. We found that Smyd2 translocated from the cytosol and Z-disc region in CTRL myocytes to the site of the titin N2A element in mdx, just as did HSP90. Thus, Smyd2 might guide HSP90 to the titin filaments in the sarcomeres, where the chaperone would then exert protective functions on the N2A element and possibly, other I-band proteins. In conclusion, while the functional consequences of the translocation of HSP90 to elastic titin in hereditary myopathies remain speculative, this chaperone binds to sub-sarcomeric sites other than those that associate with sHSPs, which implicates different protective functions.