Decreased expression of myotonic dystrophy protein kinase and disorganization of sarcoplasmic reticulum in skeletal muscle of myotonic dystrophy

Journal of the Neurological Sciences 162 (1999) 38–50

Decreased expression of myotonic dystrophy protein kinase and disorganization of sarcoplasmic reticulum in skeletal muscle of myotonic dystrophy Hideho Ueda a , Masatake Shimokawa b , Masahiko Yamamoto b , Noriyoshi Kameda b , Hidehiro Mizusawa b , Takeshi Baba a , Nobuo Terada a , Yasuhisa Fujii a , Shinichi Ohno a , 1 ,c b, Shoichi Ishiura , Takayoshi Kobayashi * b

a Department of Anatomy, Yamanashi Medical University, Yamanashi 409 -38, Japan Department of Neurology, Tokyo Medical and Dental University School of Medicine, Tokyo 113 -8519, Japan c Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113 -0032, Japan

Received 10 June 1998; received in revised form 18 September 1998; accepted 25 September 1998

Abstract Pathological expression of myotonic dystrophy protein kinase (DMPK) in skeletal muscle of myotonic dystrophy (DM) was studied by Western blot analysis, immunohistochemistry, and immunoelectron microscopy of DMPK. Western blot analysis showed that DMPK protein in DM skeletal muscles dramatically decreased. DMPK-positive muscle fibers showed typical DM pathological changes such as type I atrophy, central nuclei, nuclear chains, and sarcoplasmic masses. In degenerated DMPK-positive muscle fibers, cross-striated bands disappeared, and irregular granular DMPK-positive materials appeared in sarcoplasm. By immunoelectron microscopy, DMPK was localized in the terminal cisternae of the sarcoplasmic reticulum (SR) in DM muscle. Swollen DMPK-positive SRs were detected between well preserved myofibrils in the early stage of DM muscle degeneration, and degenerated intramembranous structures with DMPK and an accumulation of mitochondria were observed between disorganized myofibrils in degenerated DM muscle. We concluded that SR is the primary site of the degeneration of DM skeletal muscle and that the decreased DMPK might cause dysregulation of intracellular calcium metabolism, which is followed by DM muscle degeneration.  1999 Elsevier Science B.V. All rights reserved. Keywords: Myotonic dystrophy; Myotonic dystrophy protein kinase; Sarcoplasmic reticulum; Western blotting; Immunohistochemistry; Ultrastructure

1. Introduction Myotonic dystrophy (DM) is an autosomal dominant inherited disease with multisystemic disorders including myotonia and muscle weakness, cardiac conduction defects, cataracts, and premature balding [14]. To date the lengthening of tracts of trinucleotide repeats has been

*Corresponding author. Tel.: 181-3-5803-5235; fax: 181-3-58030169; e-mail: [email protected] 1 Present address: Department of Life Sciences, Graduate School of Arts and Science, The University of Tokyo, Tokyo 153-8902, Japan.

recognized as a major cause of more than 12 human genetic diseases, including fragile X syndromes, Huntington’s disease, X-linked spinal and bulbar muscular dystrophy, spinocerebellar ataxia types 1, 3, and 6, Friedreich’s ataxia, dentatorubral-pallidoluysian atrophy and DM [1,7,12]. In the last 10 years, it has been reported that the DM gene localizes on chromosome 19q13.3 [6,17] and has a mutational expansion of a repetitive trinucleotide sequence (CTG)n located in a 39 noncoding region of the myotonic dystrophy protein kinase (DMPK) gene [11,27]. A characteristic genetic feature of DM is increasing disease severity and earlier onset from generation to generation, which is called genetic anticipation [5,13,41].

0022-510X / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0022-510X( 98 )00290-1

H. Ueda et al. / Journal of the Neurological Sciences 162 (1999) 38 – 50

The DM gene product: DMPK has a strong amino acid homology to members of the protein kinase family [5]. We recently discovered DMPK as a transmembrane protein that is located mainly in the terminal cisternae of the sarcoplasmic reticulum (SR) using immunoelectron microscopy [35] and an antibody produced against the DMPK C-terminus [33]. Immunocytochemically, DMPK is localized in type I muscle fibers but is not colocalized with SERCA II ATPase, which is localized primarily in the longitudinal SR [22]. To understand the pathophysiological basis of DM muscle degeneration, we studied the quantity of DMPK in DM skeletal muscles by Western blot analysis and compared the localization of DMPK in DM skeletal muscles with well-known DM pathological changes using histological, histochemical and immunocytochemical techniques, confocal laser scanning microscopy, and immunoelectron microscopy.

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sequence corresponding to amino acid residues (SGAAQEPPALPEP) of the human DMPK C-terminus [33]. The peptide-specific antibody was purified from antisera using an affinity column (Pharmacia Biotech, Tokyo, Japan) according to the manufacturer’s protocol [35]. To investigate the pathological changes in DMPK localization at the light microscopy level, we used monoclonal antibodies against human slow myosin heavy chain (MHC), which recognizes type I muscle fiber, and against human fast MHC, which recognizes type IIA, IIB, and IIC muscle fibers ( [18]; these were generous gifts from Prof. T. Shimizu, Department of Neurology, Teikyo University), and a monoclonal anti-SERCA II ATPase antibody (MA3910, Affinity Bioreagents, Inc., Neshanic Station, NJ, USA), which strongly immunostained the longitudinal SR of the type I (slow) muscle fibers.

2.3. Western blot analysis 2. Materials and methods

Human skeletal muscle biopsies were homogenized in phosphate buffered saline (PBS) and centrifuged at 10 000 g for 15 min. The supernatant was used as the soluble fraction. The pellet was suspended in a buffer (10% sodium dodecyl sulfate (SDS), 0.125 M Tris-HCl, pH 6.8, 30% glycerol, 5% 2-mercaptoethanol and 0.02% bromophenol blue), boiled for 5 min at 1008C and centrifuged at 10 000 g for 15 min. The supernatant was used as the membrane-rich fraction. All buffers contained 1 mmol / l phenylmethylsulfonyl fluoride and 1 mg / ml each of pepstatin A and leupeptin. All procedures were performed at 48C. About 100 mg of each sample was loaded onto 9% SDS polyacrylamide gels for electrophoresis and subsequently blotted onto Immobilon polyvinylidene difloride membranes (Millipore, Bedford, MA, USA). After being blocked with Tris-buffered saline with Tween 20 (TBST) containing 5% skim milk, the membranes were incubated

2.1. Muscle preparations Skeletal muscle biopsies of four adult DM patients, aged 29 to 59, and three adults (a 22-year-old male, a 30-yearold male and a 50-year-old female) who showed no intrinsic muscle diseases as controls. Diagnosis was established by history, clinical examination, electromyogram and histological and histochemical investigations of biopsies obtained from brachioradialis, biceps, or quardriceps with informed consent (Table 1).

2.2. Antibodies The anti-DMPK antibody was produced in rabbits against a peptide synthesized from the predicted peptide

Table 1 Clinical and histological and histochemical features of patients with myotonic dystrophy Case No

Sex

Age (years)

Duration of symptoms (years)

Clinical feature

Histological and histochemical features Type I fiber atrophy

Type II fiber hypertrophy

Central nuclei and nuclear chains

Sarcoplasmic mass

Ring fiber mass

Type 1

Type 2

Type 1

Type 2

Type 1

Type 2

1

M

29

3

Muscle myotonia, distal muscle atrophy and hypotonia, hatchet face, frontal baldness, testicular atrophy

2

M

37

14

Muscle myotonia, distal muscle atrophy and hypotonia, hatchet face, cataract, diabetes mellitus

11

1

111

1

1

6

1

11

3

M

40

13

Muscle myotonia, distal muscle atrophy, cataract, conduction block

11

1

11

1

1

6

1

11

4

F

59

15

Muscle myotonia, muscle atrophy and hypotonia, hatchet face, cataract, Af

1

11

111

1

1

6

6

1

6: rare, 1: sometimes, 11: often, 111: remarkable in one specimen. We examined more than two different specimens in each case

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with a polyclonal anti-DMPK antibody in TBST overnight at 48C. After being rinsed in TBST, they were incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (Amersham, Little Chalfont, UK). They were visualized using an enhanced chemiluminescence system (Amersham) according to the manufacturer’s protocols. For the quantitative analysis of DMPK, the MHC concentration of each sample solution was pre-measured by densitometric analysis with brilliant Coomassie Bluestained 6% SDS-PAGE. Immunoblotting was done under conditions in which each sample solution had equal MHC content.

2.4. Histological, histochemical and immunohistochemical procedures To compare the localization of DMPK in DM skeletal muscle fibers with the well known pathological changes in DM skeletal muscle, we performed conventional histological, histochemical and immunohistochemical studies on semi-sequential sections. For histological and histochemical studies, frozen muscle biopsy specimens were sectioned at 7 mm thickness and mounted on poly-L-lysinecoated slides and air-dried for 30 min. All samples were stained with hematoxilin and eosin (H&E), modified Gomori trichrome, NADH-TR, myofibrillar ATPase and other stains according to standard methods [9]. For immunohistochemical studies, the cryosections were preincubated for 30 min with PBS containing 2% bovine serum albumin and 5% goat or rabbit serum (G solution). These sections were incubated with anti-DMPK, anti-slow, or anti-fast MHCs in G solution overnight at 48C. After being washed in PBS for 20 min, the sections were incubated with HRP-conjugated goat anti-rabbit immunoglobulin (IgG) (Cappel, West Chester, PA, USA) or HRP-conjugated rabbit anti-mouse IgG (Chemicon, Temecula, CA, USA). HRP-conjugates were visualized by diaminobenzidine (DAB). For the immunocontrol, PBS or normal rabbit or mouse serum was used instead of the primary antibodies.

2.5. Immunofluorescent studies of double labeling of DMPK /slow or fast MHCs and DMPK /SERCA II ATPase Frozen biopsy specimens were sectioned at 7 mm thickness, mounted on poly-L-lysine-coated slides and air dried for 30 min. The cryosections were preincubated for 30 min with PBS containing 2% BSA and G solution. The sections were incubated with anti-DMPK antibody in G solution at 48C overnight. After being washed in PBS for 20 min, the sections were incubated with rhodamineconjugated goat anti-rabbit IgG (Cappel) in G solution at room temperature for 1 h, and then washed in PBS. For double immunofluorescent labeling of DMPK / slow or fast MHCs and DMPK / SERCA II ATPase, the tissue sections

were incubated with anti-slow or -fast MHCs or SERCA II ATPase antibodies at 48C overnight, washed, and incubated with fluorescein-isothiocyanate (FITC)-labeled goat anti-mouse IgG (Cappel) at room temperature for 1 h. For the immunostain control, PBS or normal rabbit or mouse serum was used instead of the primary antibodies. After the application of coverslips, the sections were observed with a LSM 310 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

2.6. Image analysis by confocal microscopy From double-immunofluorescent-stained tissue sections, data from two channels were collected simultaneously to provide a precise colocalization and individually analyzed to show the localization of each antigen. The data from one channel are indicated in green (FITC-fluorescence: excitation, 488 nm argon laser) and the data from the other channel are indicated in red (rhodamine-fluorescence: excitation, 543 nm HeNe laser). To examine the distribution of the two antigens, data from both channels are overlaid to produce a single image on which regions of colocalization are indicated in yellow.

2.7. Immunoelectron microscopy 2.7.1. Electron microscopy of pure morphology Some DM muscle biopsies were routinely fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) for 60 min and postfixed with 1% osmium tetroxide in PB for 60 min. They were dehydrated in a series of graded ethanols and pure acetone, and embedded in Quetol 812 (Nisshin EM, Tokyo, Japan). Ultrathin sections at 70 nm thickness were counterstained with uranyl acetate and lead citrate, and observed under a H-600 or H-8100 electron microscope (Hitachi, Tokyo, Japan). 2.7.2. Pre-embedding immunoelectron microscopy Some specimens were prepared for immunoperoxidase staining with streptavidin-biotin method using Histofine kits (Nichirei Corp., Tokyo, Japan). Staining procedures were almost identical to the immunofluorescence staining until the primary antibody incubation. Subsequently, they were incubated with anti-rabbit IgG antibody conjugated to biotin for 10 min and streptavidin conjugated to HRP for 5 min, following the manufacturer’s protocol. They were fixed again with 0.25% glutaraldehyde in PB for 10 min and treated with metal-enhanced DAB (Pierce Lab., Rockford, IL, USA) for 15 min and 1% osmium tetroxide in PB for 30 min. They were dehydrated in a series of ethanols and embedded in epoxy resin by the inverted gelatin capsule method. Ultrathin sections were lightly counterstained by uranyl acetate and lead citrate and observed under an electron microscope. For the immunocontrols, we used PBS or normal rabbit serum instead of the polyclonal anti-DMPK antibody.

H. Ueda et al. / Journal of the Neurological Sciences 162 (1999) 38 – 50

2.7.3. Post-embedding immunoelectron microscopy Some biopsies were fixed with 2% paraformaldehyde in PB, pH 7.3, at 48C for 2 to 6 h and some were additionally fixed with 0.05% glutaraldehyde in PB for 1 h. They were then treated with 30% sucrose to prevent ice-crystal formation and embedded in OCT compound for freezing at 2808C. For immunogold staining, they were dehydrated in a series of graded ethanols at 2258C and embedded in LR-Gold (Polysciences, Warrington, PA, USA). They were polymerized under an ultraviolet beam at 2258C for 24 h. Ultrathin sections were placed on nickel grids. After treatment with 1% BSA for 10 min, they were incubated with anti-DMPK antibody for 2 h, and then with goat anti-rabbit IgG antibody conjugated to 5 nm-colloidal gold (Chemicon) for 2 h. They were counterstained with only uranyl acetate and observed with an electron microscope.

3. Results

3.1. Western blot analysis of DMPK Our antibody recognized a protein band of 70 kDa in the membrane-rich fraction (Fig. 1a, lanes 5–7), which corresponded to the full-length DMPK [35], and a protein band

Fig. 1. Western blot analysis of human skeletal muscle biopsies of DM patients and controls. Biopsied samples of DM patients (lanes 1–4) and controls (lanes 5–7) were analyzed by immunoblotting with anti-DMPK antibody. The controls have both 70- and 55-kDa proteins, but DM patients have dramatically decreased 70-kDa proteins and also decreased 55-kDa protein. a: membrane-rich fraction. b: soluble fraction. Lane 1: muscle from DM case 1; lane 2: muscle from DM case 2; lane 3: muscle from DM case 3; lane 4: muscle from DM case 4; lane 5: muscle from a normal 22-year-old male; lane 6: muscle from a normal 30-year-old male, and lane 5: muscle from a normal 50-year-old female.

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of 55 kDa in the soluble fraction (Fig. 1b, lanes 5–7) in the controls. In DM patients, the 70 kDa protein bands dramatically decreased and the 55 kDa protein bands also decreased (Fig. 1a and b, lanes 1–4).

3.2. Light microscopy studies of DM skeletal muscles Western blot analysis showed that DMPK protein in DM skeletal muscle dramatically decreased. Immunohistochemically, however, the expression of DMPK and its pathological changes were quite variable even in the same specimen. We chose areas with slight pathological changes for double immunolabeling studies of DMPK / MHCs and DMPK / SERCA II ATPase and those with more advanced pathological changes for comparison with the well-known DM pathological changes and the process of DM muscle degeneration.

3.2.1. Colocalization of DMPK /slow MHC and DMPK / SERCA II ATPase in DM skeletal muscle fibers In Fig. 2a–e and Fig. 3 it is demonstrated that DMPK is exclusively colocalized in slow (type I) muscle fibers and not colocalized in fast (type IIa, IIb, and IIc) muscle fibers that appear similar to normal skeletal muscle fibers [22] by H&E, myofibrillar ATPase staining, and peroxidase staining of DMPK, slow and fast MHCs on semi-serial sections (Fig. 2a–e) and by confocal scanning microscopy of the double immunolabeling of DMPK / slow or fast MHCs (Fig. 3). Type I muscle fibers that show almost the same intensity of slow MHC (Fig. 2d and Fig. 3b) have weak, moderate, and strong intensities of DMPK (Fig. 2e, 3a and d), which is similar to the controls [22]. Double immunofluorescent labeling of DMPK / SERCA II ATPase showed that DMPK-positive muscle fibers are exclusively colocalized with SERCA II ATPase-positive muscle fibers (Fig. 4a–c), but at higher magnification, DMPK-positive crossstriated bands are observed between SERCA II ATPasepositive cross-striated bands (Fig. 4d–f). 3.2.2. Comparison of DMPK localization and wellknown DM pathological changes In Table 1 the histological and histochemical features of DM skeletal muscles are summarized. Almost all angular atrophic fibers were DMPK-positive at weak or moderate intensity (Fig. 2g, Fig. 3a–c and Fig. 4a–c), and these atrophic fibers were detected from very slightly pathological lesions (Fig. 5a and b) to severely degenerated lesions. DMPK-negative (type II) muscle fibers were usually hypertrophic (Fig. 2f and g). Typical central nuclei and nuclear chains were observed mainly in DMPK-positive (type I) muscle fibers (arrowheads in Fig. 2f, g, h, and i), but also found in some DMPK-negative (type II) muscle fibers (in Fig. 2f, g, h, and i). Typical sarcoplasmic masses were observed in DMPK-positive muscle fibers (Fig. 2j and k). Interestingly, the region that appears to be homogenous in the periphery of sarcoplasm

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H. Ueda et al. / Journal of the Neurological Sciences 162 (1999) 38 – 50

Fig. 2. Histological, histochemical, and immunohistochemical photomicrographs of DM biopsied muscle in comparison with the localization of DMPK and the classical DM pathological changes. a, f, h, j, l, n and p: H&E staining, b: myofibrillary ATPase staining after pre-incubation of pH 4.5., and immunohistochemical stainings of fast MHC (c), slow MHC (d and q) and DMPK (e, g, i, k, m, o and r). DMPK localizes in type I (slow MHC-positive) muscle fibers at different intensities (strong: arrowhead, moderate: double arrowhead, and weak: triple arrowhead) but does not localize in type II (fast MHC-positive) muscle fibers. 1: type I muscle fiber, 2A and 2B: type IIA and IIB muscle fibers. Classical DM pathological changes stained with H&E are compared with peroxidase immunostaining of DMPK and slow MHC. Most muscle fibers with central nuclei (arrowheads in f) are DMPK-positive (arrowheads in g), but one muscle fiber with central nuclei (arrow in f) is DMPK-negative (arrow in g). Type II muscle fibers (DMPK-negative ones) are hypertrophic (g). Many muscle fibers with nuclear chains (arrowheads in h) are also DMPK-positive (arrowheads in i), but one muscle fiber (arrow in h) is DMPK-negative (arrow in i). A typical sarcoplasmic mass (arrowhead in j) has irregular staining of DMPK (arrowhead in k). Dense peripheral staining of DMPK (arrows in m) often appears in a homogeneous lesion of the peripheral sarcoplasm (arrows in l) in a DMPK-positive muscle fiber, which is supposed to be the very early formation of a ‘sarcoplasmic mass’. Sarcoplasmic masses are usually observed in DMPK-positive fibers. A nuclear clump (arrow in l) is DMPK-negative (arrow in m). Typical ring fibers in a DMPK-negative muscle fiber (arrowheads in n and o) and a DMPK-positive (r) type I (slow MHC-positive in q) fiber (arrowheads in p–r) are shown. Ring fibers are more common in type II muscle fibers than in type I muscle fibers. Pictures of a–e, h, and i are taken from case 3, those of f and g are taken from case 1, those of j–r are taken from case 2. Bar, 100 mm in a–i and 50 mm in j–r.

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Fig. 3. Double immunofluorescent staining of DMPK (a, d) and slow (b) and fast (e) MHCs on DM skeletal muscle. c and f: Simultaneous double-immunofluorescent pictures of DMPK / slow and fast MHCs. DMPK staining is completely negative in fast muscle fibers (d–f) and DMPK staining is exclusively colocalized in slow muscle fibers (a–c). The intensities of DMPK in slow muscle fibers are strong (arrowheads) moderate (double arrowheads), and weak (triple arrowheads). Angular atrophic fibers (arrows) have usually weak or moderate DMPK immunostaining. All pictures are taken from case 2. Bar, 500 mm.

(Fig. 2l) and which seem later to become a sarcoplasmic mass at the light microscopy level, had denser staining of DMPK (Fig. 2m) in comparison with the typical sarcoplasmic mass (Fig. 2j and k). Typical sarcoplasmic masses were rarely observed in DMPK-negative (type II) muscle fibers. Many hypertrophic ring fibers were DMPK-negative (Fig. 2n and o), but some DMPK-positive ring fibers were detected (Fig. 2p–r) in all DM muscles. Pycknotic nuclear clumps were DMPK-negative (arrow in Fig. 2l).

3.2.3. Pathological process of the DMPK localization in degenerated DM skeletal muscle fibers In lesions with early DM pathological changes, large muscle fibers of different sizes had homogeneous distribution of DMPK in each muscle fiber with staining of various intensities (Fig. 5a and b), but angular atrophic fibers had weak staining of DMPK (Fig. 5a and b). Hypercontracted fibers had strong DMPK staining in the periphery of the sarcoplasm, but it was very weak in the center (Fig. 5c and d). With progressive muscle degeneration, the regular cross-striated pattern of DMPK disappeared and irregular granular DMPK-positive materials appeared (Fig. 5e, f and g). In moderately to severely degenerated DM muscle fibers, the localization of DMPK became heterogeneous (Fig. 5e, h and i) and strongly positive DMPK-areas were often observed in the periphery of the sarcoplasm (Fig. 5e) or just underneath the plas-

malemma (Fig. 5h). DMPK-positive granules became variable in size, and DMPK-negative areas were often observed (Fig. 5h and i). In the more advanced stages, irregular DMPK-positive granules became more sparsely distributed in the sarcoplasm (Fig. 5j). Control sections showed no immunoreactivity of the DMPK.

3.3. Ultrastructural studies of DM skeletal muscles 3.3.1. Pure morphology of DM skeletal muscle Although some parts of DM skeletal muscle fibers demonstrated relatively normal ultrastructures (Fig. 6a) including the triad structure (Fig. 6a, inset), most of the muscle fibers showed a focal dilatation of intramembraneous structures (Fig. 6b) and disorganization of Z bands. In addition, the ultrastructural abnormalities of the triad were often recognized as irregular dilatations and distortions of the T-tubules, and extremely swollen SR (Fig. 6c). In more degenerated regions, myofilaments were irregularly organized, and the typical triad structure was not recognized (Fig. 6d). 3.3.2. Immunoelectron microscopical analysis of DM skeletal muscles Immunoreactivity of DMPK was recognized in the terminal cisternae of SR, even in the swollen ones, although it was sometimes difficult to distinguish the

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Fig. 4. Double immunofluorescent staining of DMPK (a, d) and SERCA II ATPase (b, e) in DM skeletal muscle. c and f: Simultaneous double immunofluorescent pictures of DMPK / SERCA II ATPase. Low power views (a–c) show that DMPK is colocalized in SERCA II ATPase-positive muscle fibers. The intensities of DMPK are strong (arrowheads), moderate (double arrowhead) and weak (triple arrowhead) in SERCA II ATPase-positive muscle fibers (a–c) similar to those in slow muscle fibers in Fig. 3. High power views show that a banding pattern of DMPK (d) is detected between a cross-striated pattern of SERCA II ATPase (e) with longitudinal string-like staining in a band and no clear colocalization of DMPK and SERCA II ATPase is observed (f). All pictures are taken from case 4. Bar, 100 mm in a–c and 50 mm in d–f.

terminal cisternae of SR from the longitudinal SR (Fig. 7a–c). In Fig. 7a relatively normal ultrastructures except for SR swelling, in which DMPK immunoreactive products are recognizable near the Z-bands are shown. The inset of Fig. 7a shows DMPK localized in the site facing T-tubules, probably indicating the junctional face of the terminal cisternae of SR. In moderately degenerated regions, most of the SR were swollen and DMPK immunoreactive products were irregularly aligned (Fig. 7b). Even in severely damaged regions, where many myofibrils were frayed and membranous structures were sparsely distributed, DMPK immunoreactivity was sometimes readily visible in swollen SRs (Fig. 7c; inset). Immunogold labeling showed DMPK recognized at the junctional face of the terminal cisternae of SR (Fig. 8a). Moreover, peripheral coupling structures had a similar DMPK immunoreactivity (Fig. 8b).

4. Discussion In the present study, Western blot analysis and immunocytochemical tests clearly demonstrated that DMPK expression in DM skeletal muscle decreases. Other papers have also reported a decrease of DMPK proteins in DM skeletal muscle [23,26,42], although some researchers reported no change [4]. Wang et al. [43] reported that

analysis of poly(A)1 RNA from classical adult-onset DM patients showed dramatic decreases of both the mutant and normal DMPK RNAs, so both normal and expanded DMPK genes are transcribed in patient skeletal muscle, but the abnormal expansion-containing RNA has a dominant effect on RNA metabolism by preventing the accumulation of poly(A)1 RNA (dominant-negative RNA mutation). Nuclear aggregation of DMPK trinucleotide repeat transcription [37], and the altered processing of DMPK transcription [25] have also been reported. If these abnormal transcriptions manifest at the RNA level in DM muscle, the expression of DMPK protein might decrease to less than half of normal muscle. Sasagawa et al. [34] also reported that CTG repeat expansion caused a decrease in the translational rate of DMPK cDNA in COS-1 cells transfected with artificial (CTG) repeat expansion. The dramatically decreased expression of DMPK protein in DM muscle is possibly due to a dominant-negative mutation at the RNA level [39,40]. Atrophy of type I muscle fibers, increased central nuclei, nuclear chains, sarcoplasmic masses, ring fibers, abnormal terminal innervation and abnormal features of muscle spindles are characteristic features of DM muscle pathology. In our studies, DMPK is exclusively localized in slow (type I) muscle fibers [22], neuromuscular junctions in vivo and in vitro [22] and in muscle spindles. Using different antibodies against DMPK, type I dominant im-

H. Ueda et al. / Journal of the Neurological Sciences 162 (1999) 38 – 50

munostaining has also been reported by van der Ven et al. [42], Salvatori et al. [32] and Dunne et al. [10]. Several researchers also reported that DMPK was localized in

45

neuromuscular junctions [10,26,36,42,44] and muscle spindles [36,42]. Our present histological, histochemical, and immunocytochemical studies on the localization of DMPK

Fig. 5. Histological and immunohistochemical staining of degenerated DM muscle fibers. a, b, e–j: immunofluorescent staining of DMPK, c: H&E staining, and d: immunoperoxidase staining of DMPK. DMPK-positive muscle fibers have various intensities (strong: arrowhead, moderate: double arrowheads and weak: triple arrowhead), whereas angular atrophic muscle fibers usually show weak or moderate staining (arrows in a and b). Hypercontracted muscle fibers (arrowheads in c, d) which appear in H&E staining have strong DMPK-positive staining in the periphery of the sarcoplasm (arrows in d) and very weak staining in the center of muscle fibers (d). A completely degenerated DMPK positive muscle fiber (*) in c and d. During the degenerative process of DM muscle fibers, DMPK staining in a muscle fiber becomes heterogeneous (e). Some areas have densely DMPK-positive staining (arrowheads in e) with various DMPK-positive granules. In longitudinal sections, granular DMPK-positive materials (arrowheads in f) appear in the region still containing cross-striated patterns (arrows in f) and in cross-sections they appear also as many granular deposits (arrow in g). In more degenerated DM muscle fibers, DMPK appear in or beneath the plasmalemma (arrow in h and arrowhead in i), DMPK-positive granular materials become more irregular and various in size (h–j), and no DMPK-positive areas are present in the sarcoplasm (curved arrows in h and i). In a more advanced stage, various DMPK-positive granules become less and sparse (j). All pictures are taken from case 3. Bar, 100 mm in a–d and 50 mm in e–j.

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Fig. 6. Electron micrographs of the pure morphology of DM muscle fibers. (a) Very early morphological changes with microvacuoles mainly in I bands and A-I junctions. Inset: a triad structure. (b) Dilated SRs (arrowheads) with moderate morphological changes are shown. (c) A distorted triad structure is shown. Dilated SR and T-tubules are recognized. (d) Severely degenerated regions show randomly organized myofilaments and degenerative structures (arrowhead). SR, sarcoplasmic reticulum. Z, Z-band. All pictures are taken from case 2. Bar, 1 mm (a–f). A bar in inset of a is 0.5 mm.

in DM skeletal muscle in comparison with classical DM pathology clearly show that well known DM pathological features such as type I muscle fiber atrophy, central nuclei, nuclear chains, and sarcoplasmic masses, with the exception of ring fiber formation, are predominantly observed in DMPK-positive (type I) muscle fibers, so the disregulation of DMPK might be responsible for the formation of pathological features in these DMPK-positive cells. Interestingly a strong DMPK-positive region appears in the homogenous area on the periphery of sarcoplasm before the formation of a typical sarcoplasmic mass. The sarcoplasmic mass may be one of the degenerative products containing DMPK-positive materials. During the process of degeneration of DM skeletal muscle, regular crossstriated bands of DMPK disappeared and DMPK-positive granular material appeared in degenerated DM muscle fibers. These degenerated muscle fibers stained heterogeneously for DMPK, and irregular DMPK-positive granules varied in size. In the advanced stages of muscle degeneration in DM, DMPK-positive granular materials are sparsely distributed in sarcoplasm. Immunoelectron microscopy studies show that the granular materials which are observed by immunofluorescent microscopy are aggregations

of DMPK-positive swollen SRs. DMPK-positive swollen SRs appear between well preserved myofibrils. These findings are consistent with earlier work of Mussini et al. [28] and the recent DMPK-knockout mouse study by Reddy et al. [29]. Mussini et al. [28] carefully selected DM patients in the very early stages of the disease process and found microvacuoles observable on the I bands level by light microscopy and considerable disorganization of the SR detectable electron microscopically. Furthermore, DMPK-knockout mice developed a late onset progressive myopathy and electron microscopically visible focal dilation of the SR, degenerating mitochondria, and focal loss of the Z band were observed [29]. Interestingly Benders et al. [3] reported that their DMPK-knockout mice had no apparent abnormality light microscopically, but their myotubes exhibited a higher resting intracellular Ca 21 ([Ca 21 ] i ) and smaller and slower Ca 21 transient due to high extracellular potassium concentration. It has been also reported that cultured DM muscle cells showed significantly higher cytoplasmic Ca 21 levels than normal muscle cells in the presence of extracellular Ca 21 , while almost no difference between them in Ca 21 -free buffer was recognized [15]. If the intracellular

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Fig. 7. Immunoelectron micrographs of DM skeletal muscle fibers immunostained with anti-DMPK antibody and visualized by HRP-DAB technique. (a) Longitudinal section with mild morphological changes. DMPK immunoreactivity is recognized in the dilated SR (arrowheads). Inset: the DMPK immunoreactivity in the SR (arrowheads) is recognized in the site facing T-tubule (T). (b) Oblique section with moderate morphological changes. Dilated irregularly swollen SRs seem to be aggregated, and in some swollen SRs, DMPK immunoreactivity is recognized (arrowheads) but has no polarity. (c) Longitudinal section with severe morphological changes. Myofilaments have become loose and frayed. Inset: more severely degenerated region. Z-lines are decreased in number and membranous organelles are sparsely distributed, but the DMPK can be still recognized. Arrowheads indicate DMPKimmunoreactive products. A: A-band, I: I-band, M: mitochondria, SR: sarcoplasmic reticulum, T: transverse tubule, and Z: Z-band. All pictures are taken from case 3. Bar, 0.5 mm.

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Fig. 8. Immunogold electron micrographs of DM skeletal muscles. (a) Relatively normal regions. Gold particles are localized at the terminal cisternae of SR, especially at the junctional face of the SR. (b) The DMPK immunoreactivity is also recognized in the sarcoplasm near the sarcolemma (S), which resembles peripheral coupling structures in muscle fibers. Arrowheads indicate the regions where many gold particles locate. M: mitochondria, N: nucleus, S: sarcolemma, SR: sarcoplamic reticulum, T: transverse tubule, Z: Z-band. All pictures are taken from case 4. Bar, 0.2 mm.

Ca 21 remains higher, it subsequently activates various proteases that induce cell death. Damiani et al. [8] reported that defective expression of the slow / cardiac isoform of Ca 21 -binding protein calsequestrin in DM skeletal muscle and DM slow-twitch muscle fibers had a maturationalrelated abnormality with or without an altered modulatory mechanism of SR Ca 21 -transport. Our studies have clarified that DMPK localizes in the terminal cisternae of both normal and DM muscles and DMPK-positive cells are susceptible to degeneration. Furthermore, the initial degenerative feature of DM skeletal muscle is the appearance of DMPK-positive swollen SRs, and the disorganized process of SR may lead to the degeneration of DM muscle. Our polyclonal antibody detected 70-kDa protein in the membrane-rich fraction and a 55-kDa protein in the soluble fraction of both normal and DM skeletal muscle. Saitoh et al. [31] reported that when a full-length DMPK was transfected into rat L6 myoblasts and about 95% of

expressed DMPK was recovered from the light microsomal fraction. Recently, Koike et al. [24] purified a native full-length DMPK from rat skeletal muscle SR. On the basis of the cDNA sequence, human DMPK was previously reported to have two amino acid sequence variations at the C-terminus. Using two specific antibodies against rat DMPK, we demonstrated that both native DMPKs had two isoforms. Therefore, it is highly possible that human DMPK has a truncated form and our 55-kDa protein may be a N-terminus truncated form of DMPK protein. Muscle myotonia is one of the main clinical features of DM. DM muscle has a low resting membrane potential [21] and an apamin receptor [30]. The combination of a decreased resting membrane potential and the presence of an apamin-sensitive Ca 21 -activated K 1 channel can create repetitive action potentials. The remaining apamin receptors, decreased resting membrane potential, decreased Na 1 / K 1 -ATPase and SR Ca 21 -ATPase activities [2] and

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polysynapse [21] may be a maturation-related disturbance of the DM membrane. DMPK-knockout mice have progressive degenerative changes in skeletal muscle but no myotonia [16,29]. Recently Klesert et al. [20] reported that the transcript levels of the DM-associated homeobox protein (DMAHP) gene, which is located in the adjacent downstream of a CTG-repeat of the 39 untranslated region of the DMPK gene, were greatly reduced from the expanded allele in comparison with those from the wildtype allele. Thornton et al. [38] also showed that DMAHP expression in myoblasts, muscle, and myocardium was reduced by the DM mutation and that the magnitude of this effect depended on the extent of the CTG repeat expansion. It is quite interesting that the DMAHP sequence is very similar to a murine transcription factor that regulates the expression of the Na 1 / K 1 ATPase a1-subunit in developing skeletal muscle [19]. Recently, proteins with binding specificity for DNA CTG repeats and RNA CUG repeats have been identified [39,40], so these DNA- or RNA-binding proteins and / or chromatin structures may alter the regulation and processing of DMPK DNA, RNAs such as DMAHP RNA, and other RNAs transcribed from ‘maturation related’ genes. In conclusion, dysregulation of DMPK might be one of the most important factors in the degeneration of DM skeletal muscle. Further studies of the localization and function of DMPK in other tissues and to investigate the role of other genes in CpG islands will clarify the molecular pathophysiological basis of the multisystem disorder in DM, leading possibly to the discovery of a fundamental treatment for DM.

Acknowledgements This work was supported in part by grants from the Ministry of Health and Welfare to S. Ishiura and T. Kobayashi and by a grant-in-aid for scientific research 07670702 from the Ministry of Education, Science, Sport and Culture to T. Kobayashi. We thank Miss Y. Kato and Miss K. Ariizumi for their excellent technical assistance.

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