Sarcoplasmic reticulum Ca2+-ATPase and acylphosphatase activities in muscle biopsies from patients with Duchenne muscular dystrophy

Clinica Chimica Acta, 158 (1986) 245-251 Elsevier

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CCA 03540

Sarcoplasmic reticulum Ca2+-ATPase and acylphosphatase activities in muscle biopsies from patients with Duchenne muscular dystrophy Nerina * Institute

Landi ‘, Paolo Nassi a,*, Gianfranco Liguri a, Susanna Cinzia Sbrilli b and Giampiero Marconi b

of Biological Chemistry, and b Department

of Neurology

Bobbi b,

University of Florence, Florence (Italy)

(Received January 26th, 1986; revision received May 12th, 1986; accepted after revision May 15th, 1986) Key words: Co ’ + -ATPase; Acylphosphatase;

Duchenne muscular dystrophy

Sarcoplasmic reticulum Ca*+-ATPase, acy 1p hosphatase and other soluble enzymes (creatine kinase, lactate dehydrogenase, aldolase and pyruvate kinase) were assayed in muscle biopsies from patients affected by Ducherme muscular dystrophy (DMD) and from normal controls. Specific activities of all the soluble enzymes were decreased in dystrophic muscle, acylphosphatase exhibiting the most marked and significant decrease, comparable to that of creatine kinase, in spite of a moderate increase in serum levels. Also, Ca*+-ATPase, particularly the calcium-dependent activity, was decreased in dystrophic muscle. A positive correlation, higher than with the other soluble enzymes, was obtained between acylphosphatase specific activity and the percentage of Ca*’ -activation of Cat+-ATPase. These findings: (i) suggest an impairment of microsomal calcium uptake which could be, at least in part, responsible for sarcoplasmic calcium accumulation observed in DMD; (ii) do not disagree with an hypothesized role of acylphosphatase in intracellular calcium homeostasis, consistent with the enzyme’s demonstrated hydrolytic activity on the phosporylated intermediate of Ca*+-ATPase.

Introduction

In Duchenne muscular dystrophy (DMD) the fundamental biochemical abnormality has not been identified. Increased intracellular calcium may be an important pathogenetic.factor being one of the earliest biochemical changes in this

* To whom correspondence and requests for reprints should be addressed biologica, Universit& di Firenze, viale Morgagni 50, 50134 Firenze, Italy.

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0 1986 Elsevier Science Publishers B.V. (Biomedical

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disorder [1,2]. However, the primary defect which might account for the rise of sarcoplasmic calcium levels is poorly understood: the influx of calcium could be secondary to a membrane defect [3,4] or the active extrusion of calcium might be decreased or the intracellular calcium transport might even be affected. With respect to the last hypothesis, calcium accumulation was found to be impaired in the sarcoplasmic reticulum (SR) fraction from biopsied muscle of patients with DMD [5], but the mechanism responsible for the deficient Ca*+ transport is yet far to be elucidated [6]. For the past few years, we have been studying functional and structural properties of acylphosphatase, purified to homogeneity from tissues of various species, including human skeletal muscle [7]. From subcellular fractionation and cytochemical studies, muscle acylphosphatase appears to be localized in the cytoplasm in proximity to the sarcoplasmic reticulum. This enzyme, which specifically catalyzes the hydrolysis of the carboxyl-phosphate bond of acylphosphates, was found to act on essential physiological substrates, such as carbamoylphosphate, 3-phosphoglyceroylphosphate, y-glutamylphosphate, /%aspartylphosphate and succinoyl-phosphate [8]. The physiological function of acylphosphatase is still being debated. It has been postulated that in active oxidizing cells, where the rate of glycolysis may be limited by low concentrations of inorganic phosphate and ADP, acylphosphatase, by hydrolyzing 3-phosphoglyceroylphosphate, may increase the rate of glycolysis at the expense of ATP formation. It is also possible that acylphosphatase, by controlling the concentration of highly reactive compounds such as acylphosphates, may prevent the acylation of proteins in tissues [9]. More recent studies from this laboratory have shown that acylphosphatase can hydrolyze the phosphorylated intermediate of Ca2+-ATPase, both in SR vesicles and in their pepsin digest [lo]. This activity suggests a possible involvement of this enzyme in intracellular calcium homeostasis, due to its regulation of the efficiency of Ca*+ uptake by sarcoplasmic reticulum. Based on the foregoing considerations, we undertook a study on the levels of SR Ca*+-ATPase, acylphosphatase and other enzymes in muscles of patients affected by DMD. Materials and methods Muscle specimens

Muscular biopsies were examined from a total of 11 individuals: 6 patients affected by muscular dystrophy and 5 normal controls. All patients (or their parents) gave informed consent. Biopsies of quadriceps (vastus lateralis) muscle were taken under local anesthesia with lidocaine or general anesthesia with ketamine hydrochloride (Ketalar, Parke-Davis, Laimate, Milan), care being taken not to infiltrate the muscle. One clamped specimen was divided into several pieces which were immediately frozen in isopenthane cooled by liquid nitrogen for morphological and biochemical studies.

241

~ystrophic

patients

Five Duchenne muscular dystrophy patients (ranging in age from 3 to 5 yr) and one Becker dystrophy patient (aged 12 yr) were studied. Diagnoses were based on typical clinical, electromyographic and histological criteria and serum enzyme levels. All patients were ambulatory and showed comparable degrees of muscular impairment. In terms of the functional classification of Archibald and Vignos ill] they fell into the first or second class. Normal controls

Normal quadriceps muscular biopsies were taken from 5 young men (ranging in age from 15 to 21 yr), undergoing orthopaedic surgery for the correction of traumatic fractures. The control group showed neither clinical signs nor laboratory findings of muscle disease. Histological

studies

For histological studies, the following staining methods were used: EmatoxilineEosine, Gomori, Van Gieson, Weigert, Sudan Black, PAS, NADH tetrazolium reductase, alkaline and acid ATPase and acid phosphatase. Biochemical

studies

All the following operations were carried out at 0-4°C. Muscle samples, weighing about 150-200 mg, were homogenized for 120 s in 10 vol. of 75 mmol/l Tris-HCl buffer, pH 7.4, containing 0.15 mol/l KCI, using a 2 ml Waring Biendor homogenizer. The homogenate was centrifuged for 20 mm at 10000 x g in a Spinco Beckman centrifuge. The resulting supematant was filtered through glass wool and centrifuged for 45 mm at 50000 X g in the same centrifuge. This supematant was used to determine the activities of the cytoplasmic enzymes. The pellet at 50000 X g was used to prepare sarcoplasmic reticulum vesicles (SRV) according to the method of Weber et al, slightly modified 1121. The final sediment, suspended to a protein con~ntration of about 10 mg/ml, was stored at 4°C and used within 24 h for ATPase activity determination. Soluble enzyme assays The enzymatic activities for creatine kinase, lactate dehydrogenase,

aldolase and pyruvate kinase were determined by continuous optical tests at 340 nm 113-151. Assays were performed at 25OC, in a Model 550 S Perkin-Elmer spectrophotometer. Acylphosphatase was assayed continuously at 283 nm, with benzoylphosphate as substrate. The unit of activity is defined as the amount of the enzyme that liberates 1 pm01 of benzoate/min at 25°C and at pH 5.3 [16]. Acylphosphatase was also assayed, by the same procedure, in serum of dystrophic patients and normal controls. Protein determination

Soluble enzyme activity in the 50000 X g supematant was referred to non-collagen protein, estimated by incubating the samples overnight with 0.05 mol/l

248

NaOH at room temperature; after centrifugation for 15 min at 2000 x g and at room temperature, the protein content was determined by the biuret method of Beisenhertz et al [17]. The same calorimetric procedure, without NaOH treatment, was followed to determine SRV protein concentration. A TPase assay

ATPase activity was determined by a coupled optical test at 340 nm and 37°C. The assay mixture contained 2.0 mmol/l ATP, 0.5 mmol/l phosphoenolpyruvate, 0.15 mmol/l NADH, 7.5 I.U. lactate dehydrogenase, 5.3 I.U. pyruvate kinase, 5.0 mmol/l MgCl,, 0.1 mmol/l CaCl, and 0.1 mol/l Tris-HCI buffer, pH 7.2, in a final volume of 1 ml. Ca2+-independent (basal) ATPase was determined under the same conditions, but without the addition of Ca” and in the presence of 1.0 mmol/l EGTA. The reaction was started by the addition pf 20 pg of SRV proteins. Statistical

analysis

The statistical significance of the data was assessed by Student’s t test. The correlation coefficient r was obtained by regression analysis. Results and discussion Morphological studies confirmed the clinical diagnosis of muscular dystrophy, showing typical features of this pathological process. The histological pattern was characterized in all cases by good conservation of the general architecture of muscle bundles, mild proliferation of endomysial and perimysial connective tissue and absence of adipose tissue replacement. There was an increased variability in the size of fibers, but the mean value of fiber diameters was generally normal or slightly higher than normal. Hypotrophic and hypertrophic fibers were not numerous; necrosis with phagocytosis was very rare in four cases while in two others (cases 1 and 4) it was more abundant. Cellular infiltration was slight or completely absent. In the histochemical study, many fibers could not be clearly differentiated by the ATPase reaction. A comparison of the specific morphological changes in the different cases showed a substantial uniformity of the pathological picture. In conclusion, it was evident that in all patients the dystrophic process was at a similar and rather early stage. The data reported in Table I show that the specific activities of all soluble enzymes tested, expressed as units per mg of non-collagen protein, are significantly decreased in dystrophic muscle. These data are consistent with the findings of other investigators and may be ascribed to a pathological release of enzymes from diseased muscle [4]. However, it should also be noted that acylphosphatase is the enzyme which exhibits the most marked and the most significant decrease ( p c 0.01) in dystrophic muscle, comparable to that of creatine kinase, in spite of a moderate increase in serum levels which were, meanly 1.3 U/ml in dystrophic patients and 0.4 U/ml in normal controls. Sarcoplasmic reticulum Ca2+-ATPase was assayed both in the presence of 0.1 mmol/l Ca” and without the addition of calcium, but with 1

249 TABLE I Enzyme levels in muscle of patients affected by Duchenne dystrophy Case number

1 2 3 4 5 6 mean SD

Controls (a = 5) Mean SD

Age (yr) 5 4 5 3 11 4

Non-coIIagen protein (m&3 muscle)

Enzyme activities (units/mg non-collagen protein) CK LDH ALD

PK

AcPase

96 104 110 110 90 110

0.14 0.91 1.06 0.95 1.02 1.21

0.50 0.41 0.35 0.37 0.30 0.36

0.28 0.25 0.18 0.28 0.27 0.27

0.44 0.30 0.38 0.35 0.13 0.35

0.31 0.30 0.36 0.36 0.23 0.27

103 8

0.98 0.15

0.38 0.07

0.26 0.04

0.32 0.11

0.30 0.05

143 16

2.02 0.17

0.51 0.10

0.31 0.03

0.47 0.05

0.65 0.02

p < 0.01

p < 0.01

p c 0.05

p < 0.05

p < 0.05

p < 0.01

CK, creatine kinase; LDH, lactate dehydrogenase; acylphosphatase.

ALD, aldolase; PK, pyruvate kinase; AcPase,

mmol/l EGTA in order to determine the ‘basal’ and the calcium-dependent activities. The results presented in Table II reveal a considerable and highly significant (p -c 0.01) decrease of Ca *+-ATPase activity in all dystrophic muscle specimens. The decrease was particularly pronounced for the calcium-dependent activity which, on the average, was about 6-times lower than that of the controls.

TABLE II Ca2+-ATPase in muscles of patients affected by Duchenne dystrophy Case number

Ca2+-ATPase activity (nmol ATP/mg SRV protein) Basal

1 2 3 4 5 6 Mean SD

Controls (R = 5) Mean SD

Ca2+ activation (W)

Ca2+-activated

12.0 8.5 2.5 8.0 7.0 18.0

28.0 17.4 6.0 19.0 12.0 34.0

9.3 5.2

19.4 10.2

26.1 2.9

127.0 9.1

p < 0.01

p -=0.01

57.1 51.1 58.3 57.8 41.7 47.1

79

250

A high positive correlation was obtained between the specific activity of acylphosphatase and the percentage of CaZf activation of Ca2+-ATPase in dystrophic muscles. This correlation coefficient (r = 0.90) was higher than those obtained between the percentage of Ca*+ activation and any of the other soluble enzymes examined. The marked reduction of SR Ca2+-ATPase activity in dystrophic muscle and the enzyme’s altered kinetic properties, particularly the marked decrease of the Ca’+-dependent activity, may account for the consequent impairment of calcium uptake in sarcoplasmic reticulum. Such an effect could contribute to the characteristic increase of intracellular calcium, observed in this disease. As regards acylphosphatase, its marked and highly significant decrease in dystrophic muscle, as well as the high degree of correlation between its levels and Ca2+-activated ATPase activity, represents a novel finding among the biochemical abnormalities associated with DMD, contrasting with Kar and Pearson’s report [IS] of no significant modifications of acylphosphatase levels in the early stages of this disease. At present, it would be highly speculative to ascribe a pathogenetic significance to this finding. Our data, however, do not disagree with the tentative hypothesis postulating an involvement of acylphosphatase in the intracellular calcium transport, consistent with the enzyme’s demonstrated hydrolytic activity on the phosphoenzyme intermediate of SR Ca’+-ATPase. We think it is worthwhile, therefore, to probe more deeply into the levels and properties of acylphosphatase in dystrop~c muscle in order to obtain experimental verification of its hypothesized role in sarcoplasmic calcium homeostasis. Acknowledgements

This work was supported by Grants from the Italian Minister0 della Pubblica Istruzione and the Cons&ho Nazionale delle Ricerche. References Emery AEH, Burt D. Intra~iIular calcium in pa~og~esis and antenatal diagnosis of Duchenne muscular dystrophy. Br Med J 1980; 302: 355-357. Bertorini TE, Bhattacharya SK, Pahnieri Genaro MA, Chemey CM, Pifer D, Baker B. Muscle calcium and magnesium content in Duchenne muscular dystrophy. Neurology 1982; 32: 1088-1092. Rowland P. Biochemistry of muscle membranes in Duchenne muscular dystrophy. Muscle & Nerve 1980; 3: 3-20. Rowland P. Pathogenesis of muscular dystrophies. Arch Neurol 1976; 33: 315-319. Takagi A, Schotland DL, Rowland P. Sarcoplasmic reticulum in Duchenne muscular dystrophy. Arch Neurol 1973; 28: 380-384. Dux L, Martonosi AN. Membrane crystals of Ca2+ -ATPase in sarcoplasmic reticulum of normal and dystropbic muscle. Muscle BE Nerve 1983; 6: 556-573. Nassi P, Liguri G, Landi L, Berti A, Stefani M, Pavolini B, Ramponi G. Acylphosphatase from human skeletal muscle: purification, some properties and levels in normal and myopathic muscles. Biochem Med 1985; 34: 166-175. Berti A, Stefani M, Liguri G, Camici G, Manao G, Ramponi G. Acylphosphat~ action on dicarboxylic acylphosphates, Jtal J B&hem 1977; 26: 377-378.

251 9 Grisolia S, Hood W. Chemotropic basis for and regulation of protein turnover: an irreversible type of elastoplastic enzyme modification. In: Kun E, Grisolia S, eds. Biochemical regulatory mechanisms in eukaryotic cells. New York: John Wiley & Sons, 1972: 137-203. 10 Stefani M, Liguri G, Berti A, Nassi P, Ramponi G. Hydrolysis by horse muscle acylphosphatase of (Ca’+-Mg*+ )-ATPase phosphorylated intermediate. Arch Biochem Biophys 1981; 208: 37-41. 11 Archibald KC, Vignos PJ. Study of contractures in muscular dystrophy. Arch Phys Med 1959; 40: 150-159. 12 Nakamura J, Yuichi E, Kazuhiko K. The formation of phosphoenzyme of sarcoplasmic reticulum. Requirement for membrane-bound Ca’+. Biochim Biophys Acta 1977; 471: 260-272. 13 Forster G, Bemt E, Bergmeyer HU. Creatine kinase determination with creatine as substrate. In: Bergmeyer HU, ed. Methods of enzymatic analysis, Vol. II. New York and London: Verlag Chemie Weinheim/Academic Press, Inc., 1974: 785-788. 14 Bergmeyer HU, Bemt E. Lactate dehydrogenase UV assay with pyruvate and NADH. In: Bergmeyer Vol. II. New York and London: Verlag Chemie HU, ed. Methods of enzymatic analysis, Weinheim/Academic Press, Inc., 1974: 574-579. 15 Bergmeyer HU, Gawehn K, Granl M. Enzymes as biochemical reagents. In: Bermeyer HU, ed. Methods of enzymatic analysis, Vol. I. New York and London: Verlag Chemie Weinheim/Academic Press, Inc., 1974: 425-522. 16 Ramponi G, Treves C, Guerritore A. Continuous optical assay of acylphosphatase with benzoylphosphate as substrate. Experientia 1966; 22: 705. 17 Beisenhertz G, Boltze HG, Bucher TH, et al. Diphosphofructose-aldolase, Phosphoglyceraldehyde-dehydrogenase, Milchsaure-dehydrogenase und Pyruvat-kinase aus Kaninchen muskulatur in einem Arbeitsgang. 2 Naturforsch 1953; 8b: 555-577. 18 Kar NC, Pearson CM. Acylphosphatase in normal and diseased human muscle. Clin Chim Acta 1972; 40: 262-265.