A developmental change in the content of parvalbumin in normal and dystrophic mouse (mdx) muscle

Journal of the Neurological Sciences, 1990, 97:261-272

2 61

Elsevier JNS 03349

A developmental change in the content of parvalbumin in normal and dystrophic mouse muscle Motoki Sano

1, Tsukasa

Yokota

1, Toyoshi

(mdx)

Endo 2 and Hiroshi Tsukagoshi ~

IDepartment of Neurology, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo 113 (Japan), and 2Third Department of Internal Medicine, University of Yamanashi, Yamanashi (Japan)

(Received 28 December, 1989) (Revised, received 12 March, 1990) (Accepted 12 March, 1990)

SUMMARY A highly sensitive enzyme immunoassay for mouse parvalbumin was developed in this study. The amount of parvalbumin was determined by a sandwich enzyme immunoassay method using anti-parvalbumin IgG-coated polystyrene balls and an anti-parvalbumin Fab'-horseradish peroxidase conjugate. Parvalbumin could not be detected in normal and dystrophic skeletal muscles of newborn mice. In normal mice, it appeared in the first postnatal week and increased linearly thereafter until the 12th week in fast twitch muscle. Rapid increase in parvalbumin was seen during 3rd and 8th week. On the other hand, parvalbumin detected in the first postnatal week increased gradually, but did not yet reach the adult level at the 16th postnatal week in slow twitch muscle. In m d x mice, fast twitch muscles such as the gastrocnemius and tibialis anterior were found to contain significantly decreased amounts of parvalbumin, compared with those in control mice. In fast twitch muscle parvalbumin in m d x mice could not be detected in the newborn, increased until 4th week and thereafter did not increase as that in normal mice. In slow twitch muscle the postnatal increase in parvalbumin content was not different from that in control mice. These results suggest that the decrease in the content of parvalbumin in dystrophic muscle may contribute to the elevation of the level of sarcoplasmic free Ca 2 + and the activated Ca 2 +-dependent proteolysis.

Correspondence to." Motoki Sano, M.D., Department of Neurology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45Yushima, Bunkyo-ku,Tokyo 113, Japan.

0022-510X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

262 K e y w o r d s : P a r v a l b u m i n : mdx m o u s e ; P o s t n a t a l d e v e l o p m e n t

INTRODUCTION

Parvalbumin, one of the intracellular calcium-binding proteins, is an acidic. water-soluble low molecular weight protein (Siegel et al. 1980). Parvalbumin was reported to have been found mainly in type II (fast twitch) skeletal muscle fibers (Celio and Heizmann 1982). The concentrations of parvalbumin in various muscles are well correlated with the relaxing time in a variety of species (Heizmann et al. 1982). Therefore. parvalbumin is supposed to be a relaxing factor in fast skeletal muscles (Haiech et al. 1979~. The contents of parvalbumin were reported to be decreased in the hind limb muscles of adult dystrophic mice (C57BL/6J(dyZJ/dy2J)) compared with normal mice

(A)

1

2

(B)

3

3

2

1

Fig. l. Immunological analysis of mouse skeletal muscle proteins after separation by SDS-PAGE. (A) Mouse fast-twitch skeletal muscle was homogenized in 4 vols. 50 mM potassium phosphate buffer (pH 7.2) containing 1 mM CaC12 and 0.43 toNI phenytmethylsulfonylfluoride. The homogenate was centrifuged at 12000 × g 10 min. and the resultant supernatant was applied to lane 1. The supernatant was heated at 85 °C for 3 min. After removal of the resultant precipitate, the heat stable muscle extract was applied to lane 2. The purified parvalbumm was applied to lane 3. Proteins were separated by SDS-PAGE (0A % SDS/15 % polyacrylamide gel) and stained with 0.01 ~ Coomassie brilliant blue. The arrow indicates parvalbumin and the arrowhead dye front. (B) Material from the polyaerylamide gel was ~ransferred to nitrocellulose filter, reacted with the anti-parvalbumin serum and then stained for an ~mmunoassay as described under Materials and Methods. The antiserum only stained parvalbumm.

263 of the same age (Klug et al. 1985). Moreover, the levels of intracellular Ca 2+ are elevated in dystrophic muscles (Bertorini et al. 1982; Bodensteiner and Engel 1978; Nylen and Wrogeman 1983). Thus the reduction of parvalbumin may contribute to the elevation of the level of sarcoplasmic free Ca 2 + in dystrophic muscles. The increase in the level of free sarcoplasmic Ca2 + may be significant with respect to the activity of Ca 2 +-activated proteases, particularly as the levels of these and lysosomal proteases are elevated in dystrophic muscles (Kar and Pearson 1976, 1978; Weinstock et al. 1958). The sandwich enzyme-linked immunosorbent assay permitted quantification of parvalbumin in homogenates of developing and electrostimulated rabbit muscles (Leberer and Pette 1986). We have also recently developed a highly sensitive enzymelinked immunoassay system for rat parvalbumin (Sano et al. 1987). The present study reports on an enzyme-linked immunoassay system developed for mouse parvalbumin and on the postnatal development in the contents of parvalbumin in muscles of X-linked muscular dystrophy mice (mdx).

MATERIALSAND METHODS Parvalbumin was purified from mouse skeletal muscles by the method described previously (Endo et al. 1985). A brief description of the purification procedures follows: mouse hind leg muscle (a mixture of red and white muscle) was homogenized in 4 vols. 50 mM potassium phosphate buffer (pH 7.2) (buffer A) containing 1 mM CaC12 and 0.43 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged and the supernatant heated to 85 °C for 3 min. After removal of the precipitate, the supernatant was brought to 55 ~ (NH4)2SO 4 saturation, stirred for 30 min and then centrifuged. Next, 0.1 vols. 50~o trichloroacetic acid was added to the resultant supernatant. The centrifuged precipitate was applied to a DEAE-Sephadex A-50 column equilibrated with buffer A containing 1 mM EDTA (buffer B). The column was extensively washed with buffer B and eluted with buffer B containing 50 mM NaC1. The main peak of a high A26o/A280ratio monitored at both 260 nm and 280 nm consists of a single band as analyzed by SDS-gel electrophoresis (Fig. 1). From 20 g leg skeletal muscle, 15 mg pure parvalbumin was obtained. An enzyme immunoassay method was developed using solid phases containing immobilized anti-parvalbuminIgG and Fab'-peroxidase conjugates. The conjugates involving specific antisera were prepared as described below. Materials Bovine brain S-100 and calmodulin were prepared as previously reported (Endo et al. 1981). Animals mdx and control mice (C57BL/10Scsn) were kindly supplied by the Central Institute for Experimental Animals (Kawasaki, Japan).

264

Buffer The regularly used buffer was 10 mM sodium phosphate buffer, pH 7.0, containing 0.1 M NaC1 and 0.1 G bovine serum albumin (fraction V, Armour Pharmaceutical Co.. Kankakee, Illinois) (buffer C).

Enzymes Horseradish peroxidase (EC 1,11.1.7" grade I, lyophilized RZ = 3.01 and pepsin from porcine gastric mucosa EC 3.4.23.1 were obtained from Boehringer Mannheim GmbH (Mannheim, F.R.G.).

A nalytical procedures Double immunodiffusion of parvalbumin and its antiserum was performed on glass slides covered with 1 ~o agarose containing 0.9~o NaC1 (Endo et at. 1981). SDS-polyacrylamide gel electrophoresis was carried out in 15 ~ gels containing 0.1 ~o SDS as described by Laemmli (1970). Proteins were stained with Coomassie brilliant blue. the level of detection for protein bands being 0.3 #g/band. The samples to be applied were treated with 1 ~o SDS and 1% 2-mercaptoethanol for 2 min in a boiling water bath. Proteins were transferred to a nitrocellulose filter according to Towbin et al. (1979). Antigenic protein bands on the nitrocellulose filter paper were detected by means of an enzyme immunoassay involving sheep peroxidaseconjugated anti-rabbit IgG (Cappel, 1"500).

A ntibodies Antisera to mouse skeletal muscle parvalbumin, bovine brain S-100 and calmodulin were raised in rabbits, essentially as described by Hidaka et al. (1983). An emulsion of purified mouse skeletal muscle parvalbumin (0.2 mg) in Freund's complete adjuvant was injected at 6 sites on the back of a rabbit and the same amount of antigen was gaven as a booster injection at l-week intervals. After 2 months, the rabbits were bled and antiserum was tested by means of double immunodiffusion. IgG was prepared by fractionation of sera with ammonium sulfate followed by passage through a column of diethylaminoethyl cellulose (Whatman Chemical Separation, Ltd.. Kent, U.K.). F(ab' )2 was prepared by digestion of IgG with pepsin, and Fab' by reduction of F(ab')z with 2-mercaptoethylamine. The amounts of IgG and its fragments were calculated from the absorbance at 280 nm (Ishikawa et al. 1983).

Preparation q/"Fab' -peroxidase conjugates Anti-parvalbumin Fab' was conjugated to horseradish peroxidase using N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (Hashida et at. 1984). The bound conjugate was applied to a column of Ultrogel ACA 44 (LKB, Stockholm. Sweden) and eluted with 0.1 M sodium phosphate buffer, pH 6.5. The amount of the conjugate was calculated from the peroxidase activity (Ruan et al. t986).

265

Assaying of peroxidase activity The activity of peroxidase was assayed by fluorometry at 30 °C using 3-(p-hydroxyphenyl)propionic acid (Aldrich Chemical Co., Inc., Milwaukee, WI) as a substrate (Imagawa et al. 1983). The fluorescence intensity was measured relative to 1 mg/1 quinine in 50 mM H2SO4, at 320 nm excitation and 405 nm emission with a Hitachi F-3000 spectrophotometer.

Preparation of IgG-coatedpolystyrene balls Polystyrene balls (3.2 mM in diameter; Precision Plastic Ball Co., Chicago, IL) were coated by absorption with anti-parvalbumin IgG (0.1 mg/ml) (Ishikawa and Kato 1980).

Enzyme immunoassay Control mice (C57BL/10Scsn), newborn and 1-24 weeks old, were anesthetized with Nembutal and decapitated, and then hindlimb muscles (tibialis anterior, gastrocnemius, soleus) were removed. Muscular dystrophy (mdx) and control mice 24 weeks old were considered adult with respect to parvalbumin, because parvalbumin contents in soleus muscle reached adult levels at the 24th week of life. A muscle extract was prepared as follows. The muscles were homogenized with a glass-Teflon homogenizer in 4 vols. 50 mM sodium phosphate buffer containing 1 mM EDTA, followed by centrifugation at 12 000 rpm for 10 min. The resultant supernatant was used for the enzyme immunoassay and protein concentration determination. Aliquots, appropriately diluted with buffer C, were subjected to the immunoassay. The amount of parvalbumin was determined by a sandwich enzyme immunoassay technique, in which an anti-parvalbumin IgG-coated polystyrene ball was incubated with a diluted aliquot (50/~1) and thereafter with 10/ag of the anti-parvalbumin Fab'peroxidase conjugate as described by Ishikawa et al. (1983).

Calculation of the specific b&ding of the anti-parvalbumin Fab'-peroxidase conjugate The specific binding of the anti-parvalbumin Fab'-peroxidase conjugate in the present enzyme immunoassay was calculated by subtraction of the fluorescence intensity for peroxidase activity non-specifically bound in the absence of parvalbumin (non-specific binding) from that bound in the presence of parvalbumin.

Statistical methods All values are given as means + SD. Comparisons between 2 groups were made by one-way analysis of variance (ANOVA). Statistical significance was accepted at F-test values of P < 0.05. The group means were compared by means of Dunette's test at P < 0.05 and P < 0.01.

266 .2 I000

Q.

100 c

~g g~ ~0

~a Parvalbumin ,pg)

Fig. 2. Standard curve for parvalbuminwith the sandwich enzymeimmunoassay.The fluorescenceintensity for peroxidase activity specificallybound was calculated by subtraction of the fluorescence intensity in the absence of parvalbumin (background) from that in the presence of parvalbumin. Mouse skeletal muscle parvalbumin, O O; bovine brain calmodulin, • O; bovine brain S-100protein, A - - A . Fluorescence intensity; 100 = 1 #g/ml quinine in 0.1 N H2SOa.

RESULTS

Characterization of the an#serum against mouse skeletal muscle parvalbumin The antiserum formed precipitin lines on double immunodiffusion analysis and did not cross-react with bovine brain calmodulin or S-100 proteins, as previously reported (Endo et al. 1985). Moreover. in the immunoblotting experiment after S D S - P A G E the antiserum against mouse skeletal muscle parvalbumin stained a 13-kDa protein in whole muscle homogenates, which comigrated with purified parvalbumin (Fig. 1.). Parvalbumin levels in skeletal muscles of control mice The levels of parvalbumin were determined with the enzyme immunoassay system using anti-mouse skeletal muscle parvalbumin antibody raised in a rabbit. A typical standard curve for the parvalbumin assay is shown in Fig. 2. No cros s-reactivity between parvalbumin and bovine brain calmodulin or S- 100 protein was observed, and the minimum amount of parvatbumin that could be detected with this method was 30 pg in 50 #1 (diluted aliquot) per tube. Thus the minimum detectable concentration was 0.6 ng/ml. Linearity was obtained within a range of 0-200 ng parvalbumin/ml. When the soluble extracts of mice skeletal muscles were subjected to proper dilution and immunoassay with various sample volumes, curves parallel to the standard curve were obtained. The recovery of the antigen from muscle was more than 90% of the homogenate, and both inter- and intraassay variations were within 10%.

267 TABLE 1 THE PARVALBUMINCONTENTS DURING POSTNATAL DEVELOPMENT OF SKELETAL MUSCLES OF mdx AND CONTROL MICE Values are expressed in ng parvalbumin/mg protein of total soluble extract as means for 5 mice. Muscle

Age (week)

mdx

Control

Soleus

1 2 3 4 6 8 10 12 14 16

18.5 23.6 28.6 31.0 34.2 35.4 37.0 38.5 40.8 42.9

17.8 24.3 27.9 29.3 33.6 37.8 38.4 38.8 41.2 43.6

Tibialis anterior

1 2 3 4 6 8 10 12 14 16

3,060 5,310 7,030 8,840 9,050 8,770 8,750 8,720 8,820 8,800

5,830 7,050 7,880 9,540 12,470 14,950 16,020 17,190 16,880 16,710

The levels of parvalbumin were determined with the enzyme immunoassay in control, newborn and 1-24-week-old mice. In newborn mice, parvalbumin was not detected at all by this enzyme immunoassay system. From the 1st week, parvalbumin was detected and increased linearily thereafter until the 12 weeks in the tibialis anterior, when the protein had reached the adult mouse levels (Table 1). The rapid increase in parvalbumin was seen during the 3rd and 8th week in the tibialis anterior (Table 1, Fig. 3b). On the other hand, in the soleus muscle, parvalbumin appeared in the 1st week and increased gradually until week 16. The amount of parvalbumin in the soleus muscle had not yet reached the adult level at 16 weeks (Table 1, Fig. 3a). The protein in the soleus muscle reached the adult level at 24 weeks of age (data not shown). Parvalbumin levels in skeletal muscles of mdx mice The levels of parvalbumin were significantly decreased in fast-twitch muscles in mdx mice as compared with normal control mice (C57 BL/10ScSn) (Table 2). However, the level of parvalbumin was not decreased in slow-twitch muscle (soleus muscle) in mdx mice (Table 2).

268

5O

"4t

40 30

jf

20 -//

:= IE

T:

xl02

180

.,O

n

140

100

6O Nb

1

2

4

8

16

week

Fig. 3. Accumulation of parvalbumin (ng/mg protein) during the postnatal development of skeletal muscles of control mice. Parvalbumin levels were determined by means of the enzyme immunoassay. The means for 5 mice are indicated in the graphs. In newborn (Nb) mice, parvalbumin could not be detected in either the soleus or the tibialis anterior. (a) In the soleus muscle, detectable level of parvalbumin appeared in week 1 and increased thereafter gradually until week 16. (b) In the tibialis anterior, parvalbumin could be detected from week 1 and increased thereafter linearly until week 12, when the amount of the protein had reached the maximum level. The rapid increase in parvalbumin was seen from week 3 to 8.

TABLE 2 THE PARVALBUMIN CONTENTS IN mdx AND NORMAL CONTROL MICE The age of both groups is 24 weeks. Values are expressed in ng parvalbumin/mg protein of total soluble extract as means _+ SD (n = 10). Muscle

mdx

Control

Gastrocnemms Tibialis anterior Soleus

7120 + 1780" 8750 + 1910"* 51 ± 7

11840 +_2280 16820 _+2950 53 _~ 5

Levels of significance (one-way ANOVA with Dunette's test) are: *P < 0.05, **P < 0.01

Developmental change in parvalbumin content in skeletal muscles of mdx mice ( T a b l e 1) A d e v e l o p m e n t a l c h a n g e in t h e levels o f p a r v a l b u m i n w a s a l s o d o t e r m i n e d in dystrophic muscles with the enzyme immunoassay system. In newborn dystrophic mice, p a r v a l b u m i n w a s n o t d e t e c t e d in e i t h e r f a s t - o r s l o w - t w i t c h m u s c l e . F r o m t h e 1st till

269

fz

50 40 30 20 r.

XlO 2 -~ 100

E

a.

0

ioo' 4O

20 Nb

u

J I

i

~

i

i

I

2

4

8

16

week Fig. 4. Accumulation of parvalbumin (ng/mg protein) during the postnatal development of skeletal muscles of mdx mice. Parvalbumin levels were determined by means of the enzyme immunoassay. The means for 5 mice are indicated in the graphs. In newborn (Nb) mdx mice, parvalbumin could not be detected in either the soleus or the tibialis anterior. (a) In the soleus muscle, the increase in parvalbumin content was almost the same as in control mouse. (b) In the tibialis anterior, parvalbumin content increased until week 4, but thereafter showed almost same level as that in week 4.

the 4th week parvalbumin in dystrophic muscle increased but remained significantly lower than in normal muscle (tibialis anterior muscle) (Fig. 4b). After the 4th week, the parvalbumin content in dystrophic muscle was not remarkably increased with respect to that of control muscle until week 16. In contrast, in the soleus muscle, the developmental change of parvalbumin content was not significantly different from that in normal muscle (Fig. 4a).

DISCUSSION

Quantitative measurement of parvalbumin used to be performed by electrophoretic technique or high performance liquid chromatography (HPLC) (Heizmann et al. 1982; Klug et al. 1985; Mtlntener et al. 1985). This was because the production of an antibody and the preparation of labelled parvalbumin are difficult since parvalbumin lacks tyrosine and tryptophan residues (Endo et al. 1985; Roth 1975). The detection limit of electrophoretic method and HPLC, however, is of microgram order. Recently the quantification of rabbit parvalbumin was first etablished using enzyme immunoassay by Leberer and Pette (1986). The development of radioimmunoassay and enzyme immunoassay clarified that slow twitch muscle contained a

270 considerable amount ofparvalbumin (Endo et al. 1985; Leberer and Pette 1986). In this experiment a highly sensitive enzyme immunoassay for mouse parvalbumin was established, involving anti-parvalbumin Fab'-peroxidase conjugate showing ne cross-reactivity with other Ca 2 +-binding proteins such as calmodulin and S-100 proteins. The results obtained with this immunoassay method reconfirmed the high content of parvalbumin in fast-twitch skeletal muscles. We also detected parvalbumin in stow-twitch muscles, such as the soleus, in which the level of the protein was less than 0.5 Z, of that in fast-twitch muscles Parvalbumin in fast skeletal muscle has been assumed to serve a~ a relaxing factor, removing Ca 2 + from troponic C and then transporting the Ca 2 ~ to the sarcoplasmic reticulum (Haiech et al 1979). Recent studies have demonstrated a strong correlation between the content of parvalbumin and the relaxing speed in a variety of mammalian skeletal muscles (Heizmann et al. 1982). In this study, soleus muscle has been found to contain only a small amount of parvalbumin, which is consistent with low relaxation speed of the muscle. The contents of parvalbumin were reported to be decreased by about 40"/0 in the hindlimb muscles of adult dystrophic mice (C57BL/6J(dy2J/dy 2J ) strain) compared with in normal control mice of the same age (Klug et a1.1985). In fast-twitch muscle the concentrations of parvalbumin m m d x mice were significantly lower than those in normal mice of the same age. Physiologically, the reduction in the content o f parvalburain may be closely related to the prolonged relaxation time of dystrophic muscles (Bressler et al. 1983; Dangain and Vrbov~t 1984; Parslow and Parry 1981). Short-term low-frequency stimulation induced the decrease in parvalbumin (Leberer and Pette 1986) and the increase in free Ca 2 ~ in fast-twitch muscle (Streter et al. 1985). Therefore. the decrease in the content of parvalbumin may cause the elevation of the level of free sarcoplasmic Ca 2- and might thus result in the activation of Ca 2 + -activated neutral protease (CANP) and lysosomal proteases. Parvalbumin was not detected in newborn mice, but soon alter the levels of parvalbumin in fast-twitch muscle increased linearly until the 12th week after birth. The onset of parvalbumin synthesis appears to be correlated with the neonatal-to-adult transition of motorneuron activity (Leberer and Pette 1986; Navarrete and Vrbovfi 1983). Moreover, the expression of parvalbumin is greatly enhanced by phasic, and drastically decreased by tonic, motor neuron activity (Leberer et al. 1986). Therefore in m d x mouse, the decrease in parvalbumin may be due to immature motor neuron activity. The reduction in the content of parvalbumin may also be due to immature muscle ceils in dystrophic muscles, which regenerate after necrosis. Taken together, the regenerated muscle cells may be in an immature state with respect to the regulation of Ca-' ~

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