The localizations of myoglobin in skeletal muscle cells of patients with Duchenne muscular dystrophy (DMD), myotonic dystrophy (MyD), and amyotrophic lateral sclerosis (ALS) were studied by immunohistochemistry and immunoelectron microscopy. In normal skeletal muscle cells, myoglobin was localized mainly in the I-band region. In degenerating muscle cells of patients with DMD and MyD, myoglobin was also demonstrated in the distended lumen of the internal membrane system and in the intermyofibrillar space, through which it seemed to pass into the extracellular space. No myoglobin was detected in opaque fibers or in some of small-sized fibers in DMD muscle. In patients with ALS the staining intensities of myoglobin varied in different muscle cells, but myoglobin was restricted to the I-band region in many muscle cells. These findings suggest that changes in the localization of myoglobin in skeletal muscle cell sensitively reflect the pathologic status of muscle cells. Key words: myoglobin skeletal muscle cell immunohistochemistry immunoelectron microscopy muscular dystrophy ALS Duchenne muscular dystrophy myotonic dystrophy MUSCLE & NERVE 14:342-347 1991

LIGHT AND ELECTRON MICROSCOPIC STUDIES ON LOCALIZATION OF MYOGLOBIN IN SKELETAL MUSCLE CELLS IN NEUROMUSCULAR DISEASES HlSAOMl KAWAI, MD, TOSHlHlKO SEBE, MD, HlROSHl NISHINO, MD, YOSHlHlKO NISHIDA, MD, and SHIRO SAITO, MD

Myoglobin, a heme protein present in skeletal and cardiac muscles, receives oxygen from the blood stream and transfers it to mitochondria.' In normal skeletal muscle cell, myoglobin is located in the I-band and on the outer membrane of mitochondria, but when the muscle is damaged it leaks easily out of the muscle cells with resultant increase of its level in the blood. The changes in serum and urinary myoglobin concentrations in various cardiac and skeletal muscle disbut eases have been studied extensi~ely,~~' 2,13z * little is known about the changes in the localization of myoglobin in damaged muscle cells. This article reports the changes of myoglobin localiza-

From the First Department of Internal Medicine, School of Medicine, University of Tokushima, Tokushima, Japan. Acknowledgment: This study was supported in part by Grant No 86-02 from the National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare of Japan. Address reprint requests to Hisaomi Kawai, MD. First Department of Internal Medicine, School of Medicine, The University of Tokushima, Kuramoto-cho, Tokushima 770, Japan Accepted for publication March 24, 1990.

CCC 0148-639X/91/040342-06 $04 00 0 1991 John Wiley & Sons, Inc

342

Myoglobin Localization

tion in skeletal muscle cells from patients with Duchenne muscular dystrophy (DMD), myotonic dystrophy (MyD), and amyotrophic lateral sclerosis (ALS). MATERIALS AND METHODS

Specimens of skeletal muscle were obtained from 2 patients with DMD aged 15 and 18 years, 2 patients with MyD (52-year-old man and 64-year-old woman), and 3 patients with ALS (2 men ages 48 and 53 years, and 1 woman age 68 years). About 1 to 1.5 g of the deltoid muscle or vastus lateralis muscle was obtained by biopsy under local anesthesia. Normal skeletal muscle obtained at surgery from a patient with no neuromuscular disease was also examined as a control.

Subjects.

Fab'-horseradish peroxidase (HRP) conjugate was prepared by the maleimide method of Ishikawa et a14 as described Preparation of Fab'-HRP Conjugate.

lmmunohistochemistry. Specimens of about 3 mm x 10 mm of biopsied muscle were clamped at the resting length and immersed in cold Zambo-

MUSCLE & NERVE

ADril 1991

ni’s fixative. l 6 They were frozen in isopentane cooled with acetone and dry ice, and stored at -70°C. Sections of 8-pm thickness were cut on a cryostat and mounted on albumin-coated slides. The direct immunoperoxidase reaction was performed as described previ~usly.~ For light microscopy, the stained sections were rinsed in PBS-NRS, dehydrated in an ethanol series, and immersed in xylene. For immunoelectron microscopy, the stained sections were fixed with 2% osmium tetraoxide, dehydrated in a graded acetone series, and embedded in epon. U1trathin sections cut with a diamond knife on a microtome were observed without staining. As controls, either Fab’-HRP prepared from normal rabbit serum or PBS was applied instead of Fab’-HRP prepared from antimyoglobin antiserum.

RESULTS Normal Skeletal Muscle

Light Microscopic Observations. Brown crossstriated staining was demonstrated in all muscle fibers (Fig. 1A). No apparent difference in intensity of staining in different fibers was observed. Electron Microscopic Observations. Staining was observed on I-bands and Z-bands and on the outer membranes of mitochondria (Fig. 1B).

Weak staining was also observed on the membranes of the internal membrane system. N o staining was detected in control specimens treated with Fab’-HRP prepared from normal rabbit serum.

Duchenne Muscular Dystrophy

Light Microscopic Observations. In muscle cells with mild degenerative changes, the intensity of staining and the striated pattern were considerably well preserved, but in those with marked degenerative changes the staining was faint or not detectable (Fig. 2A). Myoglobin was not stained in the opaque fibers, except in delta-lesion areas (Fig. 2A). Electron Microscopic Observations. In muscle cells with mild changes, regions with well-preserved myoglobin staining in the I-band and regions with diminished staining were found even in single cells. But, in muscle cells with marked histologic changes and with deteriorated internal structures, the staining was weak and its localization was greatly altered. For instance, in an opaque fiber no staining was demonstrated in either the I-band or Z-band in the hypercontracted area, but some delta-lesiong areas were stained (Fig. 2B). In a myofibril that had slipped out from among normally arranged myofibrils, no staining of either the

FIGURE 1. lmmunohistochemicaldetection of myoglobin in normal skeletal muscle. (A) Light microscopic appearance. All the muscle cells show cross-striated staining (bar = 20 prn). (B) Electron microscopic appearance. I-bands and the mitochondria1outer membrane (lower arrow) are stained. The sarcoplasmic reticulum (upper arrow) is also stained (bar = 1 pm).

Myoglobin Localization

MUSCLE & NERVE

April 1991

343

FIGURE 2. lmmunohistochemical detection of myoglobin in skeletal muscle of a case of DMD. (A) Light microscopic appearance. Staining is detected only in the delta-lesion area of an opaque fiber (*) (bar = 50 pm). (B) Electron microscopic appearance. The delta-lesion (*) area is stained but not the hypercontractedregion (**) (bar = 10 pm). (C) Staining is observed both in the lumen of the sarcoplasmic reticulum and on the membrane (arrow) (bar = 1 pm).

I-band or the Z-band was detected. However, intermyofibrillar space adjacent to this myofibril appeared to be distended and stained (Fig. 2C). In addition, myoglobin staining was seen in the lumen and on the membrane of the sarcoplasmic reticulum (Fig. 2C). The mitochondria1 outer membrane was usually stained, but the staining intensities varied with grade of muscle damage. Some small-sized muscle cells with no internal structural deteriorations were stained, but others were not.

Myotonic Dystrophy

Light Microscopic Findings. Most muscle cells showed cross-striated myoglobin staining (Fig. 3A). Electron Microscopic Findings. Myoglobin staining was demonstrated mainly in the I-band and Z-band. The I-bands in the groups of atrophic myofibrils showed decreased intensity of staining, and intermyofibrillar spaces adjacent to the I-bands were stained (Fig. 3B). In some regions of the sarcoplasmic reticulum, the lumen was stained more strongly than the I-band, and membrane of the sarcoplasmic reticulum was strongly stained (Fig. 3C). Staining of regions of the I-band adjacent to the sarcoplasmic reticulum was decreased (Fig. 3C). Membranes of mitochondria were also stained in MyD muscle (Fig. 3C).

344

Myoglobin Localization

Amyotrophic Lateral Sclerosis

Light Microscopic Findings. The staining intensity varied in different muscle cells (Fig. 4A), in particular, the staining intensity of small angulated fibers varied greatly from normal to weak (Fig. 4A). Electron Microscopic Findings. The I-bands in a fiber with no distinct structural abnormality was stained similar to those in normal muscle (Fig. 4B). Mitochondria1 outer membranes were also stained. Some of small angulated fibers showed faint staining of both the I-band and Z-band. In a target fiber, no staining was seen in the central zone, but the I-band showed a staining pattern similar to normal muscle cells in the peripheral zone. The intermediate zone was stained, but the staining intensities of the I-band and A-band were similar, and wavy Z-bands in the zone showed only slight staining (Fig. 4C). Results on the staining of myoglobin in skeletal muscles from patients with DMD, MyD, and ALS are summarized in Table 1. DISCUSSION

We previously reported studies by immunohistochemical and electron microscopic techniques on the localization of myoglobin in normal human skeletal muscle cells.5 As yet, no details of subcellular localization of myoglobin in skeletal muscle

MUSCLE & NERVE

April 1991

FIGURE 3. lmmunohistochemicaldetection of myoglobin in muscle cells of a case of MyD. (A) Light microscopic appearance. Muscle fibers show cross-striated staining (bar = 50 pm). (B) and (C) Electron microscopic appearance. (B) The grouped atrophic myofibrils (Explanation see text). (C) The lumen of a T-tube and the sarcoplasmic reticulum are stained. Staining is also seen on the mitochondria1 (Mt) outer membrane (bar = 1 pm).

cells of patients with myopathies or neuropathies have been reported. In this work, using the same techniques, we examined its localization in muscle cells of patients with DMD, MyD, and ALS. In DMD muscle, a variety of intensities and patterns of staining were seen in different muscle

cells, indicating differences in the synthesis and degradation of myoglobin in different cells or differences in myoglobin leakage from these cells. Moreover, uneven staining of the I-band in a single muscle cell suggests that the lesion is not uniform throughout each cell. T h e absence of myo-

FIGURE 4. lmmunohistochemistryand immunoelectronmicroscopic localization of myoglobin in skeletal muscle cells of a case of ALS. (A) Light microscopic appearance. The staining varies with the abnormality of muscle cells. Small angulated fibers show variable staining (bar = 100 pm). (B) and (C) Electron microscopic appearance. (B) The I-band stains for myoglobin in slightly atrophic fibers (bar = 1 pm). (C) Target fiber. (For explanation see text.) (Bar = 2 pm.)

Myoglobin Localization

MUSCLE & NERVE

April 1991

345

Table 1. Localization of myoglobin in skeletal muscle cells in normal controls and patients with neuromuscular diseases demonstrated immunohistochemically and by immunoelectronmicroscopy. Localization Myofilaments I-band A-band Z-band Mitochondria Outer membrane Inner membrane Tubular system Membrane Luminal space Nuclei lntermyofibrillar space Opaque fibers

Normal

+ +

DMD

+-+

MyD

+---t

-

+

ALS

+ +

-

++ - + + - - ++ - + ++ - + +

+ -

Regenerative fibers Target fibers Small angulated fibers

*-+

+-+

f--

&

+ +-+--

+ -

-

-

+,-

Grade ofmyoglobin staining: ++, strong; -, none.

+

+.+, moderate; 2 , mild;

globin staining in opaque fibers may suggest that (as the result of hypercontraction) myoglobin in the muscle cells was squeezed out through the deteriorated cell membrane system (including the surface cell membrane and internal membrane system) into the extracellular space. Decreased staining of the I-band in myofibrils that had slipped out from among normally arranged myofibrils, and increased staining of intermyofibrillar spaces adjacent to these displaced myofibrils, and of the lumen of the sarcoplasmic reticulum suggest that myoglobin released from the I-band may pass out of the muscle cell through the altered internal membrane system and intermyofibrillar space. This idea is consistent with the report of Oguchi’’ that the T-tube and sarcoplasmic reticulum are connected in DMD muscle. Recently, Sugita et all4 using antibody against a synthetic polypeptide for a partial sequence of dystrophin, demonstrated that dystrophin is located on the surface membrane of muscle cells. Hoffman et a13 also reported the absence of dystrophin on the surface cell membrane of DMD muscle. Koenig et a16 speculate that dystrophin is a constituent of the cytoskeleton, and that a defect

346

Myoglobin Localization

in dystrophin causes secondary alterations of the surface membrane of muscle cells. These findings indicate that myoglobin may leak out of muscle cells through the cell surface membrane into the extracellular space. Our data suggest leakage- of myoglobin from muscle cell through internal membrane system, but not the leakage through cell surface membrane. The presence of 2 different types of myoglobin staining in small-sized cells, which probably are regenerating cells, can be explained by supposing that some regenerating cells have already started myoglobin synthesis whereas others have not. In MyD muscle, cross-striated staining of most muscle cells similar to the staining of normal muscle cells was seen by light microscopy. Electron microscopic examination showed staining of the I-bands of most myofibrils, but many of the changes were similar to those in DMD muscle except for the absence of opaque fibers, although the grades of change were less than in DMD. This suggests that a similar mechanism of muscle damage may be operating in MyD muscle as in DMD muscle. In ALS, the marked variety in staining intensities of the small-sized cells could be attributed to differences in the synthesis andlor degradation of myoglobin in different muscle cells. The abnormal localization of myoglobin in target fiber show different myoglobin synthesis or degeneration in the different parts of a single muscle cell with an abnormal contractile system. The variable staining of the outer membrane of the mitochondria in diseased muscle cells indicates a very close r e l a t i ~ n ”between ~ myoglobin and mitochondria. Myoglobin is a water-soluble protein and readily leaks out of damaged muscle cell. Therefore, there must be some mechanism for fixation of this protein to specific regions in normal muscle cells, and dysfunctioning of the mechanism may result in the abnormal distributions of diseased muscle cells. Changes in the localization of myoglobin in muscle cell with morphologic changes, especially with changes in the contractile system of the muscle cell, suggest a close relation between the contractile system and the mechanism for maintaining myoglobin in situ. These changes may sensitively reflect the alteration in the muscle characteristic to each disease.

MUSCLE & NERVE

April 1991

REFERENCES

1. Covell DG, Jacquez JA: Does myoglobin contribute significantly to diffusion of oxygen in red skeletal muscle? Am J Physiol 1987;252:R34 1 -R347. 2. Edwards DL, Criddle RS: T h e interaction of myoglobin with mitochondria1 structural protein. Biochemistry 1966;5:588-59 1. 3. Hoffman EP, Knudson CM, Campbell KP, Kunkel LM: Subcellular fractionation of dystrophin to the triads of skeletal muscle. Nature 1987;330:754-758. 4. Ishikawa E, Yoshitake S, Imagawa M, Sumiyoshi A: Preparation of monomeric Fab’-horseradish peroxidase conjugate using thiol groups in the hinge and its evaluation in enzyme immunoassay and immunohistochemical staining comparing with other methods. Ann NY Acad Sci 1983;420:74- 89. 5. Kawai H, Nishino H, Nishida Y, Masuda K, Saito S: Localization of myoglobin in human muscle cells by immunoelectron microscopy. Mzcscle Nerve 1987;lO:144- 149. 6. Koenig M, Monaco AP, Kunkel LM: The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 1988;53:2 19- 228. 7. Libingston DJ, McLachlan SJ, LaMar GN, Brown WD: Myoglobin: cytochrome b, interactions and the kinetic mechanism of metmyoglobin reductase. J Biol Chem 1985;260:15699- 15707. 8. Miyoshi K, Saito S, Kawai H, lwasa M, Hayashi T , Yagita M: Radioimmunoassay for human myoglobin; methods and results in patients with skeletal or myocardial disorders. J Lab Clin Med 1978;92:341-352. 9. Mokri B, Engel AG: Duchenne dystrophy: Electron microscopic findings pointing to a basic or early abnormality in

Myoglobin Localization

the plasma membrane of the muscle fiber. Neurology 1975;25:1111-1120. 10. Nishida Y, Kawai H , Nishino H: A sensitive sandwich enzyme immunoassay for human myoglobin using Fab’horseradish peroxidase conjugate: methods and results in normal subjects and patients with various diseases. Clin Chim Acta 1985;153:93- 104. 1 1 . Oguchi K, Tsukagoshi H: An electron-microscopic study of the T-system in progressive muscular dystrophy (Duchenne) using lanthanum.] Neural Sci 1988;4:161- 168. 12. Rosano TG, Kenny MA: A radioimmunoassay for human serum myoglobin: Method development and normal values. Clin Chem 1977;23:69-75. 13. Stone MJ, Willerson JT, Gomez-Sanchez CE, Waterman MR: Radioimmunoassay of myoglobin in human serum. Results in patients with acute myocardial infarction. J Clin Invest 1975;56: 1334- 1339. 14. Sugita H , Arahara K, Ishiguro T, Suhara Y, Tsubokura T, Ishiura S, Eguchi C, Nonaka I, Ozawa E: Negative immunostaining of Duchenne muscular dystrophy (DMD) and mdx muscle surface membrane with antibody against synthetic peptide fragment predicted from DMD cDNA. Proc Japan Acad 1988;64(Ser B):37-39. 15. Sylven C, Jansson E, Szamosi A, Book K: Key enzymes of myocardial energy metabolism in papillary muscle of patients with mitral valve disease- Relation to left ventricular function. Scand J Thorac Cardiouasc Surg 1989;23:6367. 16. Zamboni L, DeMartino C: Buffered picric acidd-formaldehyde: a new rapid fixative for electron microscopy. J Cell Biol 1967;25:148A.

MUSCLE & NERVE

April 1991

347

Light and electron microscopic studies on localization of myoglobin in skeletal muscle cells in neuromuscular diseases.

The localizations of myoglobin in skeletal muscle cells of patients with Duchenne muscular dystrophy (DMD), myotonic dystrophy (MyD), and amyotrophic ...
627KB Sizes 0 Downloads 0 Views