In rats with phosphoryl-creatine depletion (fed a standard Randoin-Causeret diet 
containing 1% beta-guanidine propionic acid) abnormal mitochondria were observed 
in slow skeletal muscles, often containing paracrystalline inclusions very like
those induced by ischaemia or mitochondrial poisons and in human mitochondrial
myopathy

BERGAMINI, ETTORE

DE TATA, VINCENZO

GORI, ZINA

POLLERA, MARIA

English

Archivio della Ricerca - Università di Pisa

Br. J. exp. Path. (I988) 69, 639-650Mitochondrial myopathy in rats fed with a dietcontaining beta-guanidine propionic acid, an inhibitorof creatine entry in muscle cellsZ. Gori, V. De Tata, M. Pollera and E. BergaminiIstituto di Patologia Generale, II Cattedra, Via Roma 55, I-56 1oo Pisa, ItalyReceived for publication i I December I987Accepted for publication 6 April I988Summary. In rats with phosphoryl-creatine depletion (fed a standard Randoin-Causeret dietcontaining i% beta-guanidine propionic acid) abnormal mitochondria were observed in slowskeletal muscles, often containing paracrystalline inclusions very like those induced byischaemia or mitochondrial poisons and in human mitochondrial myopathy.Keywords: mitochondrial myopathyBeta-guanidine propionic acid (GPA) is asynthetic analogue of creatine that is readilyabsorbed at the intestinal level and theninhibits the entry ofcreatine into muscle cellsand causes depletion of creatine and creatinephosphate from muscle (Fitch et al. 1974).The addition of GPA to diet leads to thereplacement of creatine and creatine phos-phate by GPA and GPA-phosphate and mayimpair functioning of the creatine phosphateenergy-shuttle (Bessman & Carpenter I 98 5).It is well known that GPA administrationto experimental animals may lead to changesin metabolism (e.g. Fitch et al. I974; 1975),function (Fitch et al. I975; 1978; Petrofsky &Fitch I980) and structure (Schields et al.1975; Laskowski et al. I98I) of the skeletalmuscle. In this report, we show that aprolonged administration of the drug causesmitochondrial alteration in rat muscle fibres(but not in capillary endothelia) very likethat induced experimentally by ischaemia(Hanzlikova & Schiaffino I 9 7 7; Heine &Shaeg I979) or by exposing animal musclesto certain mitochondrial poisons (Melmed etal. I975; Sahgaletal. 1979) and described inhuman mitochondrial myopathy (see e.g.Scarlato & Cerri I983).Materials and methodsAnimalsRandom groups of male Sprague-Dawleyrats (ioo g initial body weight) were fed adlibitum either with a standard Randoin-Causeret diet in pellets (manufactured byPiccioni, Bergamo, Italy) or the same dietcontaining I% GPA. Addition of the drug todiet had no significant effect on food intake ordaily body weight gain (Schields & Whitehair1973) and had a significant effect on muscleweight gain very lately (Table i). Treatedanimals appeared to be healthy on casualinspection and exhibited a barely detectabledecrease in the running performance (as inSchields & Whitehair 19 73; Laskowsky et al.I98I).Correspondence: Professor Ettore Bergamini, Istituto di Patologia Generale, II Cattedra, Via Roma 5 5,56I00 Pisa, Italy.639Z. Gori et al.Table i. Effect of the administration of a diet containing I% GPA for 2, 4 and8 weeks on body weight gain and on the weight gain of the extensordigitorum longus (EDL) and of the soleus muscles (S) of ratTreatment GPA Body weight (g) EDL (mg) S (mg)2 weeks - 154±7.8 (9) 73±6.3 (9) 60±4.3 (9)+ 157±5.9 (IO) 69±3.5 (IO) 56±3.7 (IO)4 weeks - 178±7.0 (7) 82±5.7 (7) 66±5.2 (7)+ I83±iIO.6 (7) 73 ± 5.6 (7) 62± 5.3 (7)8 weeks - 360±13.6 (io) ii9±6.3 (IO) ioi±6.7 (IO)+ 258±I2.8 (io) 96±5.0 (IO)* 76±2.4 (IO)**Means ± SEM are given, in parentheses the number of cases. * and ** denotestatistically significant differences (P<0.05 and P<O.OI respectively,Student's t-test).Ultrastructural studiesOn days 7, 14, 28 and 56 after the beginningof the administration of the drug, fast (theextensor digitorum longus, EDL) and slow(the soleus, S) muscles were rapidly removedunder nembutal anaesthesia (5 mg/ioo gb.W.) and samples processed for electronmicroscopy. Small blocks of tissue fixed incacodylate-buffered 2.5% glutaraldehydewere washed in O.I M phosphate buffer (pH7.3) and post-fixed on o. I% osmium tetrox-ide in o.I M cacodylate buffer (pH 7.3),dehydrated and finally embedded in Polybed.Sections were stained with uranyl acetateand lead citrate and observed with a SiemensElmiskop I microscope.Biochemical methodsMuscles were quickly frozen with metalclamps precooled in liquid nitrogen andstored at - 70°C. The frozen muscles werehomogenized in o.6 M HC104 (5 volumes)and centrifuged at IOOO g for IO min. Thesupernatant was neutralized with KHCO32 M and centrifuged to remove the KC104sediment. All the steps were carried out ato°C. Creatine and phosphoryl-creatine wereassayed by the colorimetric procedure de-scribed by Ennor & Rosenberg (I952). GPAand phosphorylated GPA were assayedaccording to Fitch and Chevly (I975) byusing the colorimetric reaction described byBonas et al. (I963).ChemicalsGPA was synthesized and purified accordingto Rowley et al. (I 9 7 I) and incorporated intothe diet. All products used were of analyticalgrade.ResultsTable i shows that GPA administrationeventually leads to hypertrophy of both thefast-twitch (EDL) and the slow-twitch (S)muscles. Table 2 shows that this treatmentcauses a similar depletion in phosphoryl-creatine in the two muscles.At the electron microscope level, mito-chondrial alterations were observed in thesubsarcolemmal and perinuclear areas oftheS, but not of the EDL muscle; the intermyo-fibrillar mitochondria retaining a nearly nor-mal appearance. The mitochondrial changesvaried with the time of feeding. By week two(Fig. ia, b, c) pleomorphic and enlarged andgiant mitochondria (6- to 8-fold larger thannormal) were seen, sometimes with a verydense matrix and no visible cristae. Cristae640Mitochondrial myopathy in ratsTable 2. Effect ofthe administration of a diet containing I% GPA for 2, 4 and 8 weekson the content in creatine (C), phosphorylcreatine (PC), GPA and phosphoryl GPA(PGPA) of the extensor digitorum longus (EDL) and of the soleus muscles (S) of ratTreatment GPA C PC GPA PGPAEDL2 weeks - 37±2.9 (5) 39±i.6 (5)+ I7±2.7 (4)** 20±2.6 (5)** i0±0.6(4) i6±i.9 (4)4 weeks - 30±2.1 (3) 24±3.8 (3)+ io±o.8 (5)** 6± i.o (5) II±0.4(5) I7±i.6 (5)8 weeks - 28±1.9(4) 25±I.I (3)+ Io±0.4 (6)** 6±o.9 (6)** II±0.5(6) 20±0.7 (6)S2 weeks - 32±4.2 (5) 29±3.2(5)+ 19+3.3 (6)* I5±2.7 (6)** 14+1.3(3) 15±2-3 (3)4 weeks - 23± I.2(5) I4±3.3 (3)+ 7±o.8 (5)** 4±0.4 (5)* I±50.5(5) 19±2.6 (5)8 weeks - 21±I.7(4) I7±2.1 (3)+ 8±o.5 (6)** 2±0.4 (6)** 13±0.3 (6) I4±0.7 (6)Results are given as umoles/g wet muscle. Means± SEM are given, in parenthesesthe number of cases. * and ** denote statistically significant differences (P< 0.0 5 andP<o.oi respectively, Student's t-test).with an irregular orientation and with aconcentric configuration could be detectedfrequently. By week 4 (Fig. 2a, b, c), inaddition to the above-mentioned alterations,abnormal cristae with a tubular instead ofthe usual plate-like configuration wereobserved and paracrystalline inclusionscould be detected in some small, or normal orgiant mitochondria, resembling those de-scribed in experimental and human mito-chondrial myopathies. On week 8 (Fig. 3a, b),additional alterations were very elongatedforms, and enlargement with a dense matrixand cristae with an irregular often longitudi-nal arrangement (Fig. 4a, b). We saw alsovery thin and elongated worm-like orga-nelles crowded with cristae showing a regu-lar longitudinal orientation (Fig. 5a, b). Para-crystalline inclusions were seen in manymitochondria in a highly variable number(Fig. sb).Perhaps we should mention that abnor-mal mitochondria were embedded in a glyco-gen-rich sarcoplasm, and that clusters ofglycogen granules often were seen includedin round-shaped areas, which may not showa limiting membrane and that autophagicvacuoles containing membranous andamorphous material could be observed inthese mitochondria-rich areas.Signs of mitochondrial alterations werenot detectable in the EDL muscle, with theexception of an enlargement of subsarcolem-mal mitochondria (on weeks 4 and 8 oftreatment). On the other hand, heart mito-chondria were clearly altered in 8-weektreated rats (but never exhibited paracrystal-line inclusions) irrespective of their locationwithin the cell (Figs 6a, b; 7). They were quitepleomorphic, sometimes with a dense matrixand poorly defined cristae or with a verydense matrix and parallel irregularlyoriented cristae and well-defined clearintracrystal spaces, or with an increasednumber of cristae of irregular or concentricorientation. Differences in appearance were64IFig. ii. Mitochondrial alterations in the soleus muscle after two weeks ofbeta-GPA administration: cristaeappear to be increased in number and irregularly oriented; the mitochondrial matrix is electron-dense;giant mitochondria are often seen in the subsarcolemmal area, x I 5 700.AtI.,IiIFig. 2. Soleus muscle. Four weeks of beta-GPA administration. a, Enlarged mitochondia are a frequentfeature, x 6400. b, A giant mitochondrion whose cristae are irregularly oriented, x 9600. c, Sometimesthe cristae exhibit a tubular-like configuration, x 6400. d, Paracrystalline structures (arrows) arepresent in many mitochondria, x 9600.striking, and mitochondria very close to each detected (Fig. 7). Mitochondria were seenother were in a very different stage of which contain small clusters of osmiophiliccondensation. Round-shaped giant mito- lipid droplets (Fig. 6a). However, paracrystal-chondria (I0-15 gm in diameter) were line inclusions were not detectable.Fig. 3. Soleus muscle. Eight weeks of beta-GPA administration. a, Paracrystaline structures are afrequent feature (arrows). Cristae are very large and parallely oriented, x 9600. b, Cluster ofmitochondria. Paracrystalline inclusions are frequently seen in giant mitochondria, X 12 8oo.Z. Gori et al.644Mitochondrial myopathy in ratsFig. 4. Soleus muscle. Eight weeks of beta-GPA administration. a and b, Many mitochondria showparacrystalline structures (arrows), x 9600.DiscussionOur results show that beta-GPA administra-tion has a remarkable effect on the rat, sinceit induces selectively a typical mitochondrialmyopathy in the slow muscles and causesrelatively minor ultrastructural abnormali-ties in the fast muscle and in myocardialtissue. Laskowsky et al. (i 98I) could observeonly minor signs of mitochondrial alteration(dilation) in the pectoralis and gastrocne-mius muscles of chicks fed a similar diet.Since creatine depletion was similar in thetwo types of muscle, differences in the reac-tion to poison perhaps should be attributed tothe properties of type i fibres themselves,that use larger amounts of oxidative energy(e.g. Lehninger I975). Incidentally, Hanzlik-ova & Schiaffino (I977) observed a mito-chondrial myopathy in the soleus muscle ofrat after sublethal ischaemia. GPA adminis-tration (Fitch et al. 19 74; 19 75; and unpub-lished) and ischaemia (Gaja et al. I973)decrease the levels of adenine nucleotides incells, due to leakage and/or degradation bythe AMP-metabolizing enzymes. This mightbe the right perspective to understand whyGPA causes mitochondrial myopathy in typei fibres only, which contract at a higherfrequency and use a larger amount ofenergyper unit of time and have a shorter percent-age time for recovery in comparison withtype 2 and heart muscle fibres. Mitochon-drial myopathy has been observed also inhuman muscle fibres (SheHens & OssentjoukI969; Antoci & Pizzolitto I984) and appearsto be quite rare in heart muscle cells(Huebner & Grantzow I983).645'ig. 5. Soleus muscle. Eight weeks of beta-GPA administration. a and b, Peculiar worm-like)aracrystalline structures are frequently seen, either free in the cytoplasm or inside the mitochondria,x I8 400.Fig. 6. Heart muscle. Eight weeks of beta-GPA administration. Masses of pleomorphic mitochondriashowing parallel irregularly oriented cristae and a dense matrix. a, One mitochondrion contains a smallcluster of osmiophilic lipid droplets. a, x I 3 8oo; b, x 9200.Z. Gori et al.Fig. 7. Heart muscle. Eight weeks ofbeta-GPA administration. Giant and pleomorphic mitochondria withan increased number of cristae of irregular or concentric orientation, x 4600.648Mitochondrial myopathy in rats 649Our observations invite comment withregard to the pathogenesis of mitochondrialmyopathy. It has been shown that thismyopathy can be induced by many differentexperimental procedures, all of which affectthe production of energy by mitochondria:ischemia (Hanzlikova & Schiaffino I 9 7 7);anoxia (Heine & Shaeg I979); and the acuteadministration ofuncouplers of the oxidativephosphorylation (Melmed et al. I975). Inthis respect, our model appears to be differentin that there is an extramitochondrial bio-chemical lesion: the alteration ofthe creatinephosphate-dependent ATP buffering capa-city of muscle fibres, and of the transport ofenergy from mitochondria - the sites ofenergy production - to myofibrils (Bessman& Carpenter I985).Therefore our data rule out the previoushypothesis that the appearance of rod-likeintramitochondrial structures is an ex-pression of cellular hypoxia (as suggested byHeine & Schaeg 1979) and may ratherindicate that changes in energy metabolismand transfer are the true pathogenetic fac-tors of this myopathy. Because these mito-chondrial changes are similar to those seenin human mitochondrial myopathies, per-haps it should be stressed that defects increatine phosphate might have a role in thepathogenesis of the human disease.As an additional comment, there is evi-dence that treatments may not unbalancethe energy metabolism of the muscle fibressignificantly: muscle glycogen levels areincreased in GPA given rats (Baruffi et al.I984; Shoubridge et al. I985; Shoubridge &Radda I987) very like the cases of post-ischaemic experimental mitochondrial myo-pathy (Bergamini et al. 1977; Hanzlikova &Schiaffino 1977) and of the human disease(Schmalbruch I984). Perhaps the decreasein the ATP buffering capacity may lead totransient depletions of energy during con-traction and stimulate an adaptive responsein mitochondria, secondary to an increasedprotein turnover; inclusions may be of pro-teinous nature (Hanzlikova & SchiaffinoI977).AcknowledgementsThis research was supported by grants fromConsiglio Nazionale delle Ricerche (Roma)and from Ministero della Pubblica Istruzione(fondi 60% and 40%).ReferencesANTOCI B. & PIZZOLITTO S. (I984) Aspetti istoenzi-matici ed ultrastrutturali della miopatia mito-condriale (mitocondriopatia). Pathologica 76,655-668.BARUFFI F., DE TATA V., GORI Z. & BERGAMINI E.(I984) Effetti dell'intossicazione con acido-beta-guanidino propionico (APG) sul metabo-lismo del glicogeno nel muscolo scheletrico delratto. Atti 53.0 Congresso Soc. Ital. Biol. Sper.,Albamo Terme, p. 38 (Abstract).BERGAMINI E., HANZLIKOVA V. & SCHIAFFINO S.(1977) Glycogen changes in the ischemicmuscle of rat. Abstr. iith Meet. Fed. Eur.Biochem. Soc. 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Mitochondrial myopathy in rats fed with a diet containing beta-guanidine
propionic acid, an inhibitor of creatine entry in muscle cells.

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Mitochondrial myopathy in rats fed with a diet containing beta-guanidine propionic acid, an inhibitor of creatine entry in muscle cells.

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