Canliovasciilur Research, 1979, 13, 260-268
Myocardial subcellular fractionation studies on cardiomyopathic Syrian hamsters D. H . F I T C H E T T , J . S C O T T , H . R . S T E P H E N S , A N D T . J . P E T E R S ' From the Department of Medicine and Jerry Lewis Muscle Research Centre, Royal Postgraduate Medical School, London
The cardiomyopathic Syrian hamster has been the object of many studies using both biochemical and morphological techniques. Individual studies have reported a variety of abnormalities in certain subcellular organelles. It has however, been difficult to evaluate these results as each investigator has concentrated his efforts on a particular organelle, usually at the exclusion of others. In addition, several investigators have studied animals with overt heart failure and have not clearly distinguished between any primary defect and abnormalities secondary to the heart failure. The approach of analytical subcellular fractionation (de Duve, 1964,1972) separates the organelles of a particular tissue, usually by centrifugation techniques, and compares the changes in physical and enzymic properties of the various organelles in control and diseased tissue (Peters, 1977). In this manner, quantitative changes in all the subcellular organelles in the tissue can be determined. The present investigation applied this approach to a study of cardiomyopathic hamsters in animals which have no evidence of ventricular hypertrophy or overt failure and identifies a significant abnormality in an enzyme activity and centrifugal properties of the sarcolemma membrane. 'Address for reprints: DrT.J. Peters, Division of Clinical Cell Biology, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ.
Materials and methods
Male hamsters of the B10 14.6 myopathic strain and random-bred control Syrian golden hamsters were studied at between 50 and 70 days of age. The animals were anaesthetised with ether and the hearts removed. After excisting atrial tissue the ventricular muscle was homogenised and centrifuged as described previously (Bloomtield et al., 1977). Density gradient centrifugation and outer layering techniques using the Beaufay (1966) automatic zonal rotor were described by Peters and de Duve (1974). ANALYTICAL TECHNIQUES
Catalase, N-acetyl-fl-glucosaminidase, acid phosphates and neutral a-glucosidase were assayed as described by Bloomfield et al. (1977). 5'-nucleotidase was assayed according to Seymour and Peters (1977). Cathepsin D was measured by the method of Roth et al. (1971), succinate dehydrogenase was assayed as described by Prosper0 (1974) and lactate dehydrogenase according to Reeves and Fimognari (1966). Protein content of the homogenates was estimated by the Lowry (1951) technique with bovine serum albumin as standard. Enzyme activities, except for cathepsin D (Roth et al., 1971), are expressed as mUmg-' protein where I mU corresponds to the hydrolysis of 1 nmol of substrate per min.
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SUMMARY Myocardial homogenates from control animals and from hamsters with hereditary cardiomyopathy were subjected to analytical subcellular fractionation and enzymic microanalysis. Animals without ventricular hypertrophy or overt heart failure were used in this study. The principal subcellular organelles were characterised by density gradient centrifugation. Apart from evidence of enhanced lysosomal and peroxisomal fragility, probably secondary to the intracellular oedema, the intracellular organelles investigated in this study were unaffected by the myopathic process. Highly significant increases in 5'-nucleotidase activity, a marker for the sarcolemma, and an increased equilibrium density of this organelle were found in the myopathic tissue. Ultrastructural studies revealed patchy myocytolysis associated with lysosomes and with more extensive invaginations of the sarcolemma. It is suggested that a primary defect in membrane composition, leading to increased cation permeability, is the underlying abnormality in the myopathic hamster.
Organelle pathology of cardiomyopathy
261
POLYACRYLAMIDE DISC GEL ELECTROPHORESIS
Results
This was performed by the method of Maestracci et al. (1973) on the microsomal pellet, which had been boiled for 3 min in 2 % sodium dodecyl sulphate and 1% mercaptoethanol. Gels were fixed in 12.5 % trichloroacetic acid, stained with Coomassie brilliant blue and scanned at 565 nm with a Joyce Loebl scanning densitometer. The microsomal pellet was prepared as described previously (Bloomlield e t al., 1977) and has been shown to consist predominately of sarcolemma fragments.
ENZYME ACTIVITIES
LIGHT AND ELECTRON MICROSCOPY
SUBCELLULAR FRACTIONATION EXPERIMENTS
Fig. 1 shows the density gradient distribution data for the principal marker enzymes in fractionated homogenates from the control animals. Acid phosphates and catalase have predominately soluble
Table Enzyme activities and percentage activity in post-nuclear supernatant.
Enzynw and location
Lactate dehydrogenase (cytosol) 5'-nucleotidase (Sarcolemma) Neutral a-glucosidase (Endoplasmic reticulum) Succinate dehydrogenase (Mitochondria) Acid phosphatase (Lysosomes) N-acetyl-0-glucosaminidase (Lysosomes) Cathepsin D (Lysosomes) Catalase (Peroxisomes) Latent N-acetyl-0glucosaminidase (Lysosomal integrity) Protein ( m g g ' wet weight tissue)
Control Specific activity
Percentage activity in PNS fraction
3400 1 8 9 0
96
Myopathic Specific activity
Percentage activity in PNS ,fraction
E.C. No
1.1.1.27
3.29 f0.30
97 P> 0.05 62 P< 0.005 41 P> 0.05 34 P> 0.05 58
P>
P>
3010f1300
P> 0.05 19.1 f 4 . 4
44
0.160 f0.040
37
18.7 f 4 . 0
42
3.66 10.90 0.200 f0.030
65 51
15.2 f 3 . 2
51
6.30 f 1.40
62
25.0 f 4 . 2 %
-
12.9 f 0 . 3
-
31.7 f6.0 PC 0.001 0.140 fO.020 P> 0.05 17.8 f4.5
P>
0.05 0.05
0.220 f0.030 18.2 f 2 . 7 P> 0.05 7.60 f0.60
P>
P> 0.05
0.05
0.05
29.1 f8.0%
P>
0.05
12.4 f0.2 P> 0.05
3.2.1.20 I J.99.1
3.1.3.2
0.05
53 P> 0.05 57 P t 0.05 52
P>
3.1.3.5
3.2.1.30 3.4.23.5 1.11.1.6
-
-
-
-
Enzyme activities expressed as m U m g ' protein. Cathepsin D expressed in units defined by Roth et a/. (1971). Results show mean M E for 7 animals in each group.
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The animals were perfused through the left ventricle (Colgan et al., 1978) with 0.15 mol.litre-' NaCl and then with a fixative solution containing 3% glutaraldehyde, 2 % formaldehyde and 5 % dimethylsulphoxide. Small wedges of left ventricular tissue were post-fixed in 1 % osmium tetroxide, dehydrated through a graded series of ethanol and propylene oxide and embedded in Epon. Thick sections (1 pm) were stained with toluidine blue for light microscopy and thin sections were stained with lead citrate and uranyl acetate before examination in a Siemens 101 electron microscope.
The Table shows the activities of the various marker enzymes in the heart muscle homogenates from control and myopathic hamsters. The proportion of activity in the 8000 g-min (post-nuclear) supernatant in the two groups is also listed. Except for 5'-nucleotidase there was no difference between the two animal groups. The sarcolemma marker enzyme shows a 2-fold increase in activity in the myopathic group and more activity is found in the post-nuclear supernatant in the case of the myopathic than in control animals. The pH optimum for 5'-nucleotidase (9.0) was identical for both control and myopathic tissues. The apparent Km values for adenosine monophosphate were not different between the control (40 pmol.litre-l) and the myopathic (33 pmol.litre-l) enzyme activities.
D. H . Fitchett, J. Scott, H. R. Stephens, and T. J. Peters
262
Fig. 2 compares the density gradient distributions and the relative specific activity for these enzymes in myopathic tissue extracts with those from control animals. For N-acetyl-P-glucosaminidaseand catalase there is a larger propxtion of soluble activity but, with the exception of 5'-nucleotidase, the various marker enzymes show no differences between the two groups of animals. 5'-Nucleotidase shows increased levels of enzyme activity and, in addition, the median density of the particulate component is higher in the myopathic animals than in the controls.
1.15
1.25 1.05 Density
115
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1-05
1.25
Fig. I Isopycnic centrflugation (inner layering) of post-nuclear supernatant from myocardial homogenate. Frequency-density histograms ( ISD)are shown for principal marker enzymes. Frequency is defined as the fraction of total recovered activity present in the individual gradient fraction divided by the density span covered. The activity, over the abscissa interval 1.05 to 1.10, represents enzyme remaining in the sample layer of the gradient and is presumed to reflect soluble activity. The percentage recoveries ( fSD)for 3 experiments are: acidphosphatase 107fl1,cathepsin D 71 I 16, catalase 74 f12, N-acetyl-P-glucosaminidase 86 f4, 5'-nucleotidase 119 I17, neutral a-glucosidase 108 f8, succinate dehydrogenase 95+ 11, lactate dehydrogenase 84 & 9.
;];;A,In,,
30 Succimle
dehydrogemse
,
,
0 1.05
localisations with small particulate components at densities of 1.16 and 1.21 respectively. Cathepsin D and N-acetyl-P-glucosaminidase have particulate components with equilibrium densities of 1.19 although N-acetyl-fbglucosaminidase has a large proportion of soluble activity. 5'-nucleotidase shows a modal density of 1.1 1 but the peak is not separated from the original sample layer. Neutral a-glucosidase has a particulate component at a density of 1.14 and a large cytosolic component. Succinate dehydrogenase shows a symmetrical peak at a density of 1.17 but lactate dehydrogenase is almost exclusively recovered in the soluble fraction.
LCC-'~
1.15
1.25 1.05 Density
115
1.25
Fig. 2 Isopycnic centrifugation (inner layering) of post-nuclear supernatant from myocardial homogenates from myopathic (-) and control animals (..-.*). Relative frequency is obtained by multiplying the frequency for a particular fraction by the relative specific activity of the enzyme in the myopathic heart compared with the control. This factor is shown between parentheses for each enzyme. Other details as Fig. 1. The percentage recoveries ( & SD)for 3 experiments are: acid phosphatase 104 *8, cathepsin D 101 M , neutral a-glucosidase 101 f 13. 5'-nucleotidase 102i8, catalase 97 f7,succinate dehydrocenase I07 It 9, lactate dehydrocenase 94 +8.
Organelle pathology of carilioniyopathy
263
Fig. 3 shows the results of subjecting the cardiac extracts to subcellular fractionation by a flotation technique. This procedure has been claimed to enhance the separation of the various organelles from rat heart muscle (Wheeldon and Gan, 1971) although Bloomfield et a/. (1977) studying guinea pig 5' Nucleolidose CONTROL
N Acelyl-~-glucosominidose
& d i m Density llzo
POLYACRYLAMIDE GEL E L E C T R O P H O R E S I S (Fig. 4)
There were no major qualitative differences of the rnicrosomal (sarcolemma) proteins between myopathic and control animals.
la
Mellian Density 1 I53
1
-
LIGHT AND ELECTRON MICROSCOPY
01 Density
Fig. 3 Isopycnic centrifugation (outer layering) of post-nuclear supernatant fraction from myocardial homogenates from control or myopathic hamsters. Details as Fig. 1. The percentage recoveries (controll myopathic) are: 5'-nucleotidase, 81 192; N-acetyl-Pglucosaminidase. 88 189.
myocardium found that certain organelles were disrupted by the hypertonic sucrose and the resolution of the organelles was not improved. In the present study the flotation technique was used to investigate specifically the density distribution of the sarcolemma. The more usual sedimentation technique is not suitable for organelles with significant proportions of particles with equilibrium densities of less than 1.10 (Peters and de Duve, 1974). It is clear that for the myopathic extract both the median and modal densities of particulate 5'-nucleotidase are greater than in the control animal. In contrast, the distributions of the lysosomal marker enzyme N-acetyl8-glucosaminidase are identical in the two animals. These organelles are well separated by the flotation procedures.
Examination of cardiomyopathic hearts in the 60 day old animals showed focal lesions with myolysis and oedema. There was also more variability in fibre size and orientation than in the controls. With the electron microscope the alignment of the myofibrils was more often in disarray in areas of vacuolat ion and oedema. The sarcolemma infolding was more prominent in damaged fibres (Fig. 5b). The disintegration of myofilaments was associated with the presence of lysosomes (Fig. 5c) although there did not appear to be a marked increase in these organelles in less damaged fibres. Mitochondria were unaltered in the diseased hearts except for the occasional crystalline inclusion. The sarcoplasmic reticulum, particularly the 'T' tubules, was dilated in some damaged fibres (Fig. 5b). Peroxisomes were rarely seen in both normal and myopathic tissue. Discussion
The Syrian hereditary cardiomyopathic hamster provides a reliable animal model of myocardial disease. The strain used in the present study develops myocardial lesions with cellular oedema but does not progress to overt heart fai!ure for several months (Paterson et a/., 1972). The animals were studied before the development of these complications,
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10
Fig. 4 Densitometer scan of protein ban& on polyacrylamide gel stained with Coomassie brilliant blue. N o major differences exist between myopathic animals (a) and controls (b). Minor differences in peak height and R f values are apparent.
264
D. H.Fitchett, J. Scott, H. R. Stephens, and T. J. Peters
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Fig. 5A Normal lefr ventricle. This longitudinal section shows the interposition of mitochondria amongst myofibrils . A ‘T’tubule is indicated ( x 1oooO). Fig. 5B Cardiomyopathichearf. The alignment of the fibrils is less organised and sarcolemmal invaginations into the depth of the jibre are prominent (arrow). The ‘T’tubules are dilated (Double arrow) ( X 8000). Fig. 5C Oblique section of cardiomyopathic ventricle. Prominent Iysosomes are seen where tnyofihrillor rli.sinte~ration appeam to he taking place ( X 18000).
Organelle puthologv of cardiomyopathy
The discussion will consider the properties and alterations in the principal subcellular organelles under the appropriate sub-headings. SARCOLEMMA (PLASMA MEMBRANE)
fractions from myopathic tissue were due to lysosoma1 contamination of the sarcoplasmic reticulum. These workers treated their tissue homogenates with 0.6 molalitre-' KCI and this may have caused agglutination of the organelles (Bloomfield et al., 1977). In the present study the lysosomes are clearly resolved from the sarcolemma. The biochemical results presumably reflect the morphological findings of a more extensively scalloped sarcolemma. Although an undulating plasma membrane can be seen in normal fibres, the infoldings are more pronounced in association with areas of myofibrillary loss (Jasmin and Bajusz, 1975; Ferrans et a/., 1976). This may produce an increased surface membrane area. Significant alterations in extracellular space and dilated sarcoplasmic reticulum are a feature of older diseased animals (Lazarus et al., 1976) but an increase in sarcotubular membrane surface area has been reported in cardiomyopathic hearts in animals 16 to 30 days old (Colgan et al., 1978). Changes in the composition and function of the sarcolemma and the 'T' system would be expected to cause an imbalance in the excitation-contraction coupling in the myocardial cell. Excessive intracellular calcium accumulation is an important factor in the development of the cell damage in myopathic hamsters (Fleckenstein et al., 1975; McBroom and Welty, 1977). Verapamil, a drug which inhibits calcium uptake by the myocardial cell, has been shown to prevent certain histopathological abnormalities in the cardiomyopathic hamster (Jasmin and Bajusz, 1975). The direct consequences of elevated activities of 5'-nucleotidase are varied. This enzyme has been implicated in the hydrolysis of RNA (Yannarell and Aronson, 1973), the regulation of glycolysis by controlling the concentration of AMP in the cell (Atkinson, 1966; Opie et al., 1971) and affects ATP degradation (Dunnick et a/., 1972). Adenosine, the product of its action, is a potent vasodilator (Berne, 1964) and has a marked inhibitory role on ADPmediated platelet aggregation (Born and Cross, 1963). Thus 5'-nucleotidase, acting in concert with adenosine diphosphatase, may have anti-thrombotic actions (Lieberman et al., 1977). Myocardial 5'-nucleotidase itself is strongly inhibited by ATP (Ki= 1.8 Izmol.litre-') and elevated levels of the enzyme might be expected to enhance adenosine formation (Baer et d.,1966). ENDOPLASMIC RETICULUM
Although only a single enzyme (neutral a-glucosidase) localised to this organelle has been assayed, these measurements and the sucrose density gradient experiments indicate that there is no major involve-
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This organelle has the lowest equilibrium density in the myocardium and is characterised by its marker enzyme 5'-nucleotidase (Solyom and Trams 1972; W o o and Manery, 1975; Bloomfield et al., 1977). The activity of this enzyme was increased almost 2-fold in the myopathic tissue and the centrifugation experiments demonstrate an increased density and fragility of this organelle. Decreased levels of other enzymes localised in part to the sarcolemma have been demonstrated in myopathic hamsters with overt heart failure (Singh et al., 1975). These include Mg*+-dependent ATPase, Na+-, K+-activated Mg s+-dependent ATPase, Ca*+activated ATPase and adenyl cyclase. These studies, however, used preparative subcellular fractionation techniques to isolate sarcolemma and the present study has shown that the myopathic sarcolemma has different physical properties from that in control animals. Under these conditions analytical rather than preparative procedures should be employed (de Duve, 1972). It is of interest that dystrophic skeletal muscle has elevated sarcolemmal enzyme activities (Beckett and Bourne, 1957; Schubert et a/., 1960; Boegman, 1963; Arnold et a/., 1965; Dhalla et al., 1975; Neerunjun et al., 1978). The alterations in physical properties of the sarcolemma presumably reflect changes in chemical composition of the membrane: the increased equilibrium density could be caused by loss of lipid or increased protein or glycoprotein content of the sarcolemma. The recent observation of reduced sialyl transferase activities in dystrophic cardiac muscle (Limas and Limas, 1978) may underlie the changes in the composition of the sarcolemma. Singh et al. (1975) have found abnormal cholesterolphospholipid ratios in sarcolemma isolated from myopathic heart and have postulated an underlying abnormality in membrane lipo-proteins. It is of interest that alterations in the lipid content of rat liver plasma membrane markedly affect the 5'-nucleotidase activity (Chandrasekhara and Narayan, 1970). The myocardial microsomal pellet contains predominately sarcolemma as well as a small portion of sarcoplasmic reticulum (Bloomfield et al., 1977). Polyacrylamide gel electrophoresis did not show any significant qualitative differences in protein subunits present in the pellet although minor quantitative changes of uncertain significance were present. Owens et al. (1975) claimed that the changes in density gradient distributions of the membrane
265
266
D. H. Fitchett, J. Scott, H. R. Stephens, nnd T. J. Peters
ment of this organelle in the myopathic process. A major function of myocardial cell endoplasmic reticulum may be protein synthesis, particularly of membranous cell components (Dallner et al., 1966). Bester et al. (1973) found decreased protein synthetic activity of the non-ribosomal component of cell-free systems in cardiomyopathic animals.
oxidative phosphorylation and accumulation of calcium occur.
LYSOSOMES
MlTOCHONDRlA
PEROXISOMES
Although the full biochemical criteria for the presence of this organelle have not been satisfied (de Duve and Baudhuin. 1966). demonstration of particulate catalase in an organelle of relatively high equilibrium density suggests their presence in hamster myocardium as has been recently demonstrated in mouse heart (Herzog and Fahimi, 1974). We have observed similar organelles to those found by these workers in both normal and cardiomyopathic heart. Apart from evidence of increased peroxisomal fragility in the myopathic myocardium, no alterations in this organelle were noted. This enhanced fragility of the peroxisomes, like that of the lysosomes, may reflect the intracellular oedema of cardiomyopathy. This study would indicate that the primary defect in the Syrian myopathic hamster resides in the composition of the sarcolemma membrane. Increased permeability to ions, particularly calcium, could lead to the many pathological features of this condition with attempted compensatory responses of the subcellular organelles. Many previous studies have used animals with overt heart failure in which the adaptive changes and subsequent decompensation must have occurred and thus the nature of the underlying defect has been obscured by the biochemical epiphenomena. Further studies are of course necessary to delineate the nature of the sarcolemma defect but detailed analysis of the membrane components should prove valuable.
This organelle, which may contribute up to 50% of the bulk of myocardium (Spiro et al., 1968). was characterised by its marker enzyme succinate de- R E L E V A N C E T O H U M A N hydrogenase. No changes in the enzyme activity, C A R D I O M Y O P A T H Y equilibrium density or integrity of the mitochondria Although of scientific interest in its own right, study were found in the myopathic tissue. Defects in of the myopathic hamster should provide clues about oxidative phosphorylation have been described in the nature of the human diseases. However, biomyopathic myocardium (Schwartz et al., 1968) but chemical studies to date (Peters et al., 1976, 1977) the alterations were related to the degree of heart have shown normal levels of 5'-nucleotidase in failure induced (Lindenmayer et al., 1970). Early in endomyocardial biopsies from patients with conthe disease process the mitochondrial function gestive cardiomyopathies. Studies in these patients appears to be essentially normal (Lindenmayer eta/., suggest a defect in the mitochondria with secondary changes in the myofibrils and in the cytosol. How1970; Wrogemann et al., 1975). Further evidence against a mitochondrial defect ever, the congestive cardiomyopathies form a in the non-failing myopathic myocardium is pro- heterogeneous group of diseases and it is possible, vided by the demonstration of unchanged levels of particularly in familial cases, or in patients prelactate dehydrogenase. In human myocardium with senting during childhood, that a human counterpart mitochondrial damage, reduced levels of certain of the myopathic hamster may be identified. mitochondrial enzymes with increased levels of lactate dehydrogenase, have been demonstrated Supported by the British Heart Foundation and the (Peters et al., 1977). The present studies in the Muscular Dystrophy Association of Canada. We are grateful to Ms Gill Wells, Ms C e d e hamster would support the concept that only with the onset of cardiac failure d o secondary changes in Venne and Mr Peter White for expert technical assis-
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These organelles were characterised by their constituent enzymes N-acetyl-P-glucosaminidase,cat hepsin D and acid phosphatase. Apart from evidence that more N-acetyl-P-glucosaminidasewas found in the soluble fractions in myopathic as compared to control tissue, these organelles were relatively unaffected by the myopathic process. This result suggests that the lysosomes may be more fragile in the myopathic heart, which may be secondary to the intracellular oedema (Ricciutti, 1972; Welman and Peters, 1977; Wildenthal, 1978). Our ultrastructural studies suggest that lysosomes may be involved in myofilament loss in hamster cardiomyopathy. Biochemical studies in hypertrophic and myocardial failure have yielded conflicting results. Kottmeir and Wheat (1967). Schneider et al. (1971) and Tolnai and Beznak (1971) found elevated levels of lysosomal enzymes in stressed myocardium but Stoner et a/. (1973) were unable to confirm these results. Studies in dystrophic skeletal muscle (Kar and Pearson, 1972; Neerunjun et al., 1979) have shown increased lysosomal enzyme activities.
mitochondrial
Organelle patholory
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of car~lionryopathy
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Bloomfield, F. J., Wells, G., Welman, E , and Peters, T. J. (1977). Analytical subcellular fractionation of guinea pig myocardium. Clinical Science and Molecular Medicine,
generation. Recent Advances in Studies on Cardiac Struc-
D. H .
268 analysis of cardiac biopsy material from patients with valvular heart disease. Lancet. L 269-270. Peters, T. J., Wells, G., Oakley, C. M., Brooksby, 1. A. B., Jenkins, B. S., Webb-Peploe, M. M., and Coltart, D. J. (1977). Enzymic analysis of endomyocardial biopsy specimens from patients with cardiomyopathies. British Heart Journal, 39, 1333-1339.
Prospero, T. D. (1974). A simplified assay for succinate dehydrogenase. In Methodological Developments in Biochemistry, Vol. 4., pp. 41 1-412. Edited by E. Reid Longman: London. Reeves, W. J., and Fimognari, G. M. (1966). L-Lactate dehydrogenase: Heart (H,) In Methods in Enzymology Vol. IX, pp. 288-294. Edited by W. A. Wood, Academic Press: New York. Ricciutti, M. (1972). Lysosomes and myocardial cellular injury. American Journal of Cardiology, 30, 499-502. Roth, J. S., Losty, T., and Wierbicki, E. (1971). Assay of proteolytic enzyme activity using a “C-labelled haemoglobin. Analytical Biochemistry, 42,2 14-2 19. Schneider, F. N., Ito, Y., and Chidsey, C. A. (1971). Studies on lysosomal enzymes in normal and failing rabbit hearts. Journal of Cellular and Molecular Cardiology, 3, 173-1 78
.
Transactions of the New York Acarlrmy of Sciences, 30
(SUPPI. 11) 951-954. Seymour, C. A., and Peters, T. J. (1977). Enzyme activities in human liver biopsies. Assay methods and activities of some lysosomal and membrane-bound enzymes in control tissue and serum. Clinical Science and Molecular Medicine, 53,229-239.
Singh, J. N., Dhalla, N. S., McNamara. D. B.. Bajusz. E., and Jasmin, G. (1975). Membrane alteration in failing
J. Scott, H. R. Stephens,
and
T. J. Peters
hearts of cardioniyopathic hamsters. Recent Atlvances in Studies on Cardiac Structure and Metabolism, 6, 239-268. Solyom, A., and Trams, E. G. (1972). Enzyme markers in characterisation of isolated plasma membranes. Enzyme, 13,329-372.
Spiro, D., Spotnitz, H., and Sonnenblick, E. H. (1968). The relation of cardiac fine structure to function. In The Pathology of the Heart and Blood Vessels (3rd Edition), pp. 131-167. Edited by S. E. Gould. Thomas: Springfield. Stoner, C. D., Bishop, S. P., and Cirak, H.D. (1973). Normal a n d lysosomal enzyme levels in hypertrophied and failing dog hearts. Journal of Molecular anrl Cellular Cardiology, 5, 171-177.
Tolnai, S., and Beznak, M. (1971). Studies o n lysosomal enzyme activity in normal and hypertrophied mammalian myocardium. Journal of Molecular and Cellular Cardiology. 3, 193-208.
Welman, E., and Peters, T. J. (1977). Enhanced lysosomal fragility in the anoxic perfused guinea pig heart: effects of glucose and mannitol. Journal of Molecular and Cellular Cardiology, 9, 101-120.
Wheeldon, L. W., and Gan, K. (1971). Resolution o f fragments of plasma and sarcotubular membranes in heart muscle microsomes. Biochimica et Biophysica Acra, 233, 3748.
Wildenthal, K. (1978). Lysosomal alterations in ischaemic myocardium: result o r cause of myocellular damage. Journal of Molecular and Cellular Cardiology, 10, 595-605.
Woo, Y-T., and Manery, J . F. (1975). 5’-nucleotidase: an ectoenzyme of frog skeletal muscle. Biochimica PI Biophysira Acta, 391, 144-1 52.
Wrogernann, K., Blanchaer, M. C., Thakar, J. H., and Mezon, B. J. (1975). On the role of mitchondria in the hereditary cardiomyopathy. Recent Advances in Strrclirs in Cardiac Structure anrl Metabolism. 6, 23 1-24 I , Yannarell, A., and Aronson, N. N. (1973). Localization of an RNA-degrading system of enzymes on the rat liver plasma membrane. Biochimica ct Biophysica Acta, 31 1, 191-204.
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Schubert, J. R.,Muth, 0. H.,Oldfield, J. E.,and Remmert, L. F. (1960). Enhanced 5’-nucleotidase activity associated with white muscle disease. Proceedings of the Society of Experimental Bio1og.v and Medicine. 104, 568-569. Schwartz, A., Lindenmayer, G. E., and Harigaya. S. (1968). Respiratory control and calcium transport in heart mitochondria from the cardiomyopathic Syrian hamster.
Fitchett,
Myocardial subcellular fractionation studies on cardiomyopathic Syrian hamsters D. H . F I T C H E T T , J . S C O T T , H . R . S T E P H E N S , A N D T . J . P E T E R S ' From the Department of Medicine and Jerry Lewis Muscle Research Centre, Royal Postgraduate Medical School, London
The cardiomyopathic Syrian hamster has been the object of many studies using both biochemical and morphological techniques. Individual studies have reported a variety of abnormalities in certain subcellular organelles. It has however, been difficult to evaluate these results as each investigator has concentrated his efforts on a particular organelle, usually at the exclusion of others. In addition, several investigators have studied animals with overt heart failure and have not clearly distinguished between any primary defect and abnormalities secondary to the heart failure. The approach of analytical subcellular fractionation (de Duve, 1964,1972) separates the organelles of a particular tissue, usually by centrifugation techniques, and compares the changes in physical and enzymic properties of the various organelles in control and diseased tissue (Peters, 1977). In this manner, quantitative changes in all the subcellular organelles in the tissue can be determined. The present investigation applied this approach to a study of cardiomyopathic hamsters in animals which have no evidence of ventricular hypertrophy or overt failure and identifies a significant abnormality in an enzyme activity and centrifugal properties of the sarcolemma membrane. 'Address for reprints: DrT.J. Peters, Division of Clinical Cell Biology, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ.
Materials and methods
Male hamsters of the B10 14.6 myopathic strain and random-bred control Syrian golden hamsters were studied at between 50 and 70 days of age. The animals were anaesthetised with ether and the hearts removed. After excisting atrial tissue the ventricular muscle was homogenised and centrifuged as described previously (Bloomtield et al., 1977). Density gradient centrifugation and outer layering techniques using the Beaufay (1966) automatic zonal rotor were described by Peters and de Duve (1974). ANALYTICAL TECHNIQUES
Catalase, N-acetyl-fl-glucosaminidase, acid phosphates and neutral a-glucosidase were assayed as described by Bloomfield et al. (1977). 5'-nucleotidase was assayed according to Seymour and Peters (1977). Cathepsin D was measured by the method of Roth et al. (1971), succinate dehydrogenase was assayed as described by Prosper0 (1974) and lactate dehydrogenase according to Reeves and Fimognari (1966). Protein content of the homogenates was estimated by the Lowry (1951) technique with bovine serum albumin as standard. Enzyme activities, except for cathepsin D (Roth et al., 1971), are expressed as mUmg-' protein where I mU corresponds to the hydrolysis of 1 nmol of substrate per min.
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SUMMARY Myocardial homogenates from control animals and from hamsters with hereditary cardiomyopathy were subjected to analytical subcellular fractionation and enzymic microanalysis. Animals without ventricular hypertrophy or overt heart failure were used in this study. The principal subcellular organelles were characterised by density gradient centrifugation. Apart from evidence of enhanced lysosomal and peroxisomal fragility, probably secondary to the intracellular oedema, the intracellular organelles investigated in this study were unaffected by the myopathic process. Highly significant increases in 5'-nucleotidase activity, a marker for the sarcolemma, and an increased equilibrium density of this organelle were found in the myopathic tissue. Ultrastructural studies revealed patchy myocytolysis associated with lysosomes and with more extensive invaginations of the sarcolemma. It is suggested that a primary defect in membrane composition, leading to increased cation permeability, is the underlying abnormality in the myopathic hamster.
Organelle pathology of cardiomyopathy
261
POLYACRYLAMIDE DISC GEL ELECTROPHORESIS
Results
This was performed by the method of Maestracci et al. (1973) on the microsomal pellet, which had been boiled for 3 min in 2 % sodium dodecyl sulphate and 1% mercaptoethanol. Gels were fixed in 12.5 % trichloroacetic acid, stained with Coomassie brilliant blue and scanned at 565 nm with a Joyce Loebl scanning densitometer. The microsomal pellet was prepared as described previously (Bloomlield e t al., 1977) and has been shown to consist predominately of sarcolemma fragments.
ENZYME ACTIVITIES
LIGHT AND ELECTRON MICROSCOPY
SUBCELLULAR FRACTIONATION EXPERIMENTS
Fig. 1 shows the density gradient distribution data for the principal marker enzymes in fractionated homogenates from the control animals. Acid phosphates and catalase have predominately soluble
Table Enzyme activities and percentage activity in post-nuclear supernatant.
Enzynw and location
Lactate dehydrogenase (cytosol) 5'-nucleotidase (Sarcolemma) Neutral a-glucosidase (Endoplasmic reticulum) Succinate dehydrogenase (Mitochondria) Acid phosphatase (Lysosomes) N-acetyl-0-glucosaminidase (Lysosomes) Cathepsin D (Lysosomes) Catalase (Peroxisomes) Latent N-acetyl-0glucosaminidase (Lysosomal integrity) Protein ( m g g ' wet weight tissue)
Control Specific activity
Percentage activity in PNS fraction
3400 1 8 9 0
96
Myopathic Specific activity
Percentage activity in PNS ,fraction
E.C. No
1.1.1.27
3.29 f0.30
97 P> 0.05 62 P< 0.005 41 P> 0.05 34 P> 0.05 58
P>
P>
3010f1300
P> 0.05 19.1 f 4 . 4
44
0.160 f0.040
37
18.7 f 4 . 0
42
3.66 10.90 0.200 f0.030
65 51
15.2 f 3 . 2
51
6.30 f 1.40
62
25.0 f 4 . 2 %
-
12.9 f 0 . 3
-
31.7 f6.0 PC 0.001 0.140 fO.020 P> 0.05 17.8 f4.5
P>
0.05 0.05
0.220 f0.030 18.2 f 2 . 7 P> 0.05 7.60 f0.60
P>
P> 0.05
0.05
0.05
29.1 f8.0%
P>
0.05
12.4 f0.2 P> 0.05
3.2.1.20 I J.99.1
3.1.3.2
0.05
53 P> 0.05 57 P t 0.05 52
P>
3.1.3.5
3.2.1.30 3.4.23.5 1.11.1.6
-
-
-
-
Enzyme activities expressed as m U m g ' protein. Cathepsin D expressed in units defined by Roth et a/. (1971). Results show mean M E for 7 animals in each group.
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The animals were perfused through the left ventricle (Colgan et al., 1978) with 0.15 mol.litre-' NaCl and then with a fixative solution containing 3% glutaraldehyde, 2 % formaldehyde and 5 % dimethylsulphoxide. Small wedges of left ventricular tissue were post-fixed in 1 % osmium tetroxide, dehydrated through a graded series of ethanol and propylene oxide and embedded in Epon. Thick sections (1 pm) were stained with toluidine blue for light microscopy and thin sections were stained with lead citrate and uranyl acetate before examination in a Siemens 101 electron microscope.
The Table shows the activities of the various marker enzymes in the heart muscle homogenates from control and myopathic hamsters. The proportion of activity in the 8000 g-min (post-nuclear) supernatant in the two groups is also listed. Except for 5'-nucleotidase there was no difference between the two animal groups. The sarcolemma marker enzyme shows a 2-fold increase in activity in the myopathic group and more activity is found in the post-nuclear supernatant in the case of the myopathic than in control animals. The pH optimum for 5'-nucleotidase (9.0) was identical for both control and myopathic tissues. The apparent Km values for adenosine monophosphate were not different between the control (40 pmol.litre-l) and the myopathic (33 pmol.litre-l) enzyme activities.
D. H . Fitchett, J. Scott, H. R. Stephens, and T. J. Peters
262
Fig. 2 compares the density gradient distributions and the relative specific activity for these enzymes in myopathic tissue extracts with those from control animals. For N-acetyl-P-glucosaminidaseand catalase there is a larger propxtion of soluble activity but, with the exception of 5'-nucleotidase, the various marker enzymes show no differences between the two groups of animals. 5'-Nucleotidase shows increased levels of enzyme activity and, in addition, the median density of the particulate component is higher in the myopathic animals than in the controls.
1.15
1.25 1.05 Density
115
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1-05
1.25
Fig. I Isopycnic centrflugation (inner layering) of post-nuclear supernatant from myocardial homogenate. Frequency-density histograms ( ISD)are shown for principal marker enzymes. Frequency is defined as the fraction of total recovered activity present in the individual gradient fraction divided by the density span covered. The activity, over the abscissa interval 1.05 to 1.10, represents enzyme remaining in the sample layer of the gradient and is presumed to reflect soluble activity. The percentage recoveries ( fSD)for 3 experiments are: acidphosphatase 107fl1,cathepsin D 71 I 16, catalase 74 f12, N-acetyl-P-glucosaminidase 86 f4, 5'-nucleotidase 119 I17, neutral a-glucosidase 108 f8, succinate dehydrogenase 95+ 11, lactate dehydrogenase 84 & 9.
;];;A,In,,
30 Succimle
dehydrogemse
,
,
0 1.05
localisations with small particulate components at densities of 1.16 and 1.21 respectively. Cathepsin D and N-acetyl-P-glucosaminidase have particulate components with equilibrium densities of 1.19 although N-acetyl-fbglucosaminidase has a large proportion of soluble activity. 5'-nucleotidase shows a modal density of 1.1 1 but the peak is not separated from the original sample layer. Neutral a-glucosidase has a particulate component at a density of 1.14 and a large cytosolic component. Succinate dehydrogenase shows a symmetrical peak at a density of 1.17 but lactate dehydrogenase is almost exclusively recovered in the soluble fraction.
LCC-'~
1.15
1.25 1.05 Density
115
1.25
Fig. 2 Isopycnic centrifugation (inner layering) of post-nuclear supernatant from myocardial homogenates from myopathic (-) and control animals (..-.*). Relative frequency is obtained by multiplying the frequency for a particular fraction by the relative specific activity of the enzyme in the myopathic heart compared with the control. This factor is shown between parentheses for each enzyme. Other details as Fig. 1. The percentage recoveries ( & SD)for 3 experiments are: acid phosphatase 104 *8, cathepsin D 101 M , neutral a-glucosidase 101 f 13. 5'-nucleotidase 102i8, catalase 97 f7,succinate dehydrocenase I07 It 9, lactate dehydrocenase 94 +8.
Organelle pathology of carilioniyopathy
263
Fig. 3 shows the results of subjecting the cardiac extracts to subcellular fractionation by a flotation technique. This procedure has been claimed to enhance the separation of the various organelles from rat heart muscle (Wheeldon and Gan, 1971) although Bloomfield et a/. (1977) studying guinea pig 5' Nucleolidose CONTROL
N Acelyl-~-glucosominidose
& d i m Density llzo
POLYACRYLAMIDE GEL E L E C T R O P H O R E S I S (Fig. 4)
There were no major qualitative differences of the rnicrosomal (sarcolemma) proteins between myopathic and control animals.
la
Mellian Density 1 I53
1
-
LIGHT AND ELECTRON MICROSCOPY
01 Density
Fig. 3 Isopycnic centrifugation (outer layering) of post-nuclear supernatant fraction from myocardial homogenates from control or myopathic hamsters. Details as Fig. 1. The percentage recoveries (controll myopathic) are: 5'-nucleotidase, 81 192; N-acetyl-Pglucosaminidase. 88 189.
myocardium found that certain organelles were disrupted by the hypertonic sucrose and the resolution of the organelles was not improved. In the present study the flotation technique was used to investigate specifically the density distribution of the sarcolemma. The more usual sedimentation technique is not suitable for organelles with significant proportions of particles with equilibrium densities of less than 1.10 (Peters and de Duve, 1974). It is clear that for the myopathic extract both the median and modal densities of particulate 5'-nucleotidase are greater than in the control animal. In contrast, the distributions of the lysosomal marker enzyme N-acetyl8-glucosaminidase are identical in the two animals. These organelles are well separated by the flotation procedures.
Examination of cardiomyopathic hearts in the 60 day old animals showed focal lesions with myolysis and oedema. There was also more variability in fibre size and orientation than in the controls. With the electron microscope the alignment of the myofibrils was more often in disarray in areas of vacuolat ion and oedema. The sarcolemma infolding was more prominent in damaged fibres (Fig. 5b). The disintegration of myofilaments was associated with the presence of lysosomes (Fig. 5c) although there did not appear to be a marked increase in these organelles in less damaged fibres. Mitochondria were unaltered in the diseased hearts except for the occasional crystalline inclusion. The sarcoplasmic reticulum, particularly the 'T' tubules, was dilated in some damaged fibres (Fig. 5b). Peroxisomes were rarely seen in both normal and myopathic tissue. Discussion
The Syrian hereditary cardiomyopathic hamster provides a reliable animal model of myocardial disease. The strain used in the present study develops myocardial lesions with cellular oedema but does not progress to overt heart fai!ure for several months (Paterson et a/., 1972). The animals were studied before the development of these complications,
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10
Fig. 4 Densitometer scan of protein ban& on polyacrylamide gel stained with Coomassie brilliant blue. N o major differences exist between myopathic animals (a) and controls (b). Minor differences in peak height and R f values are apparent.
264
D. H.Fitchett, J. Scott, H. R. Stephens, and T. J. Peters
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Fig. 5A Normal lefr ventricle. This longitudinal section shows the interposition of mitochondria amongst myofibrils . A ‘T’tubule is indicated ( x 1oooO). Fig. 5B Cardiomyopathichearf. The alignment of the fibrils is less organised and sarcolemmal invaginations into the depth of the jibre are prominent (arrow). The ‘T’tubules are dilated (Double arrow) ( X 8000). Fig. 5C Oblique section of cardiomyopathic ventricle. Prominent Iysosomes are seen where tnyofihrillor rli.sinte~ration appeam to he taking place ( X 18000).
Organelle puthologv of cardiomyopathy
The discussion will consider the properties and alterations in the principal subcellular organelles under the appropriate sub-headings. SARCOLEMMA (PLASMA MEMBRANE)
fractions from myopathic tissue were due to lysosoma1 contamination of the sarcoplasmic reticulum. These workers treated their tissue homogenates with 0.6 molalitre-' KCI and this may have caused agglutination of the organelles (Bloomfield et al., 1977). In the present study the lysosomes are clearly resolved from the sarcolemma. The biochemical results presumably reflect the morphological findings of a more extensively scalloped sarcolemma. Although an undulating plasma membrane can be seen in normal fibres, the infoldings are more pronounced in association with areas of myofibrillary loss (Jasmin and Bajusz, 1975; Ferrans et a/., 1976). This may produce an increased surface membrane area. Significant alterations in extracellular space and dilated sarcoplasmic reticulum are a feature of older diseased animals (Lazarus et al., 1976) but an increase in sarcotubular membrane surface area has been reported in cardiomyopathic hearts in animals 16 to 30 days old (Colgan et al., 1978). Changes in the composition and function of the sarcolemma and the 'T' system would be expected to cause an imbalance in the excitation-contraction coupling in the myocardial cell. Excessive intracellular calcium accumulation is an important factor in the development of the cell damage in myopathic hamsters (Fleckenstein et al., 1975; McBroom and Welty, 1977). Verapamil, a drug which inhibits calcium uptake by the myocardial cell, has been shown to prevent certain histopathological abnormalities in the cardiomyopathic hamster (Jasmin and Bajusz, 1975). The direct consequences of elevated activities of 5'-nucleotidase are varied. This enzyme has been implicated in the hydrolysis of RNA (Yannarell and Aronson, 1973), the regulation of glycolysis by controlling the concentration of AMP in the cell (Atkinson, 1966; Opie et al., 1971) and affects ATP degradation (Dunnick et a/., 1972). Adenosine, the product of its action, is a potent vasodilator (Berne, 1964) and has a marked inhibitory role on ADPmediated platelet aggregation (Born and Cross, 1963). Thus 5'-nucleotidase, acting in concert with adenosine diphosphatase, may have anti-thrombotic actions (Lieberman et al., 1977). Myocardial 5'-nucleotidase itself is strongly inhibited by ATP (Ki= 1.8 Izmol.litre-') and elevated levels of the enzyme might be expected to enhance adenosine formation (Baer et d.,1966). ENDOPLASMIC RETICULUM
Although only a single enzyme (neutral a-glucosidase) localised to this organelle has been assayed, these measurements and the sucrose density gradient experiments indicate that there is no major involve-
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This organelle has the lowest equilibrium density in the myocardium and is characterised by its marker enzyme 5'-nucleotidase (Solyom and Trams 1972; W o o and Manery, 1975; Bloomfield et al., 1977). The activity of this enzyme was increased almost 2-fold in the myopathic tissue and the centrifugation experiments demonstrate an increased density and fragility of this organelle. Decreased levels of other enzymes localised in part to the sarcolemma have been demonstrated in myopathic hamsters with overt heart failure (Singh et al., 1975). These include Mg*+-dependent ATPase, Na+-, K+-activated Mg s+-dependent ATPase, Ca*+activated ATPase and adenyl cyclase. These studies, however, used preparative subcellular fractionation techniques to isolate sarcolemma and the present study has shown that the myopathic sarcolemma has different physical properties from that in control animals. Under these conditions analytical rather than preparative procedures should be employed (de Duve, 1972). It is of interest that dystrophic skeletal muscle has elevated sarcolemmal enzyme activities (Beckett and Bourne, 1957; Schubert et a/., 1960; Boegman, 1963; Arnold et a/., 1965; Dhalla et al., 1975; Neerunjun et al., 1978). The alterations in physical properties of the sarcolemma presumably reflect changes in chemical composition of the membrane: the increased equilibrium density could be caused by loss of lipid or increased protein or glycoprotein content of the sarcolemma. The recent observation of reduced sialyl transferase activities in dystrophic cardiac muscle (Limas and Limas, 1978) may underlie the changes in the composition of the sarcolemma. Singh et al. (1975) have found abnormal cholesterolphospholipid ratios in sarcolemma isolated from myopathic heart and have postulated an underlying abnormality in membrane lipo-proteins. It is of interest that alterations in the lipid content of rat liver plasma membrane markedly affect the 5'-nucleotidase activity (Chandrasekhara and Narayan, 1970). The myocardial microsomal pellet contains predominately sarcolemma as well as a small portion of sarcoplasmic reticulum (Bloomfield et al., 1977). Polyacrylamide gel electrophoresis did not show any significant qualitative differences in protein subunits present in the pellet although minor quantitative changes of uncertain significance were present. Owens et al. (1975) claimed that the changes in density gradient distributions of the membrane
265
266
D. H. Fitchett, J. Scott, H. R. Stephens, nnd T. J. Peters
ment of this organelle in the myopathic process. A major function of myocardial cell endoplasmic reticulum may be protein synthesis, particularly of membranous cell components (Dallner et al., 1966). Bester et al. (1973) found decreased protein synthetic activity of the non-ribosomal component of cell-free systems in cardiomyopathic animals.
oxidative phosphorylation and accumulation of calcium occur.
LYSOSOMES
MlTOCHONDRlA
PEROXISOMES
Although the full biochemical criteria for the presence of this organelle have not been satisfied (de Duve and Baudhuin. 1966). demonstration of particulate catalase in an organelle of relatively high equilibrium density suggests their presence in hamster myocardium as has been recently demonstrated in mouse heart (Herzog and Fahimi, 1974). We have observed similar organelles to those found by these workers in both normal and cardiomyopathic heart. Apart from evidence of increased peroxisomal fragility in the myopathic myocardium, no alterations in this organelle were noted. This enhanced fragility of the peroxisomes, like that of the lysosomes, may reflect the intracellular oedema of cardiomyopathy. This study would indicate that the primary defect in the Syrian myopathic hamster resides in the composition of the sarcolemma membrane. Increased permeability to ions, particularly calcium, could lead to the many pathological features of this condition with attempted compensatory responses of the subcellular organelles. Many previous studies have used animals with overt heart failure in which the adaptive changes and subsequent decompensation must have occurred and thus the nature of the underlying defect has been obscured by the biochemical epiphenomena. Further studies are of course necessary to delineate the nature of the sarcolemma defect but detailed analysis of the membrane components should prove valuable.
This organelle, which may contribute up to 50% of the bulk of myocardium (Spiro et al., 1968). was characterised by its marker enzyme succinate de- R E L E V A N C E T O H U M A N hydrogenase. No changes in the enzyme activity, C A R D I O M Y O P A T H Y equilibrium density or integrity of the mitochondria Although of scientific interest in its own right, study were found in the myopathic tissue. Defects in of the myopathic hamster should provide clues about oxidative phosphorylation have been described in the nature of the human diseases. However, biomyopathic myocardium (Schwartz et al., 1968) but chemical studies to date (Peters et al., 1976, 1977) the alterations were related to the degree of heart have shown normal levels of 5'-nucleotidase in failure induced (Lindenmayer et al., 1970). Early in endomyocardial biopsies from patients with conthe disease process the mitochondrial function gestive cardiomyopathies. Studies in these patients appears to be essentially normal (Lindenmayer eta/., suggest a defect in the mitochondria with secondary changes in the myofibrils and in the cytosol. How1970; Wrogemann et al., 1975). Further evidence against a mitochondrial defect ever, the congestive cardiomyopathies form a in the non-failing myopathic myocardium is pro- heterogeneous group of diseases and it is possible, vided by the demonstration of unchanged levels of particularly in familial cases, or in patients prelactate dehydrogenase. In human myocardium with senting during childhood, that a human counterpart mitochondrial damage, reduced levels of certain of the myopathic hamster may be identified. mitochondrial enzymes with increased levels of lactate dehydrogenase, have been demonstrated Supported by the British Heart Foundation and the (Peters et al., 1977). The present studies in the Muscular Dystrophy Association of Canada. We are grateful to Ms Gill Wells, Ms C e d e hamster would support the concept that only with the onset of cardiac failure d o secondary changes in Venne and Mr Peter White for expert technical assis-
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These organelles were characterised by their constituent enzymes N-acetyl-P-glucosaminidase,cat hepsin D and acid phosphatase. Apart from evidence that more N-acetyl-P-glucosaminidasewas found in the soluble fractions in myopathic as compared to control tissue, these organelles were relatively unaffected by the myopathic process. This result suggests that the lysosomes may be more fragile in the myopathic heart, which may be secondary to the intracellular oedema (Ricciutti, 1972; Welman and Peters, 1977; Wildenthal, 1978). Our ultrastructural studies suggest that lysosomes may be involved in myofilament loss in hamster cardiomyopathy. Biochemical studies in hypertrophic and myocardial failure have yielded conflicting results. Kottmeir and Wheat (1967). Schneider et al. (1971) and Tolnai and Beznak (1971) found elevated levels of lysosomal enzymes in stressed myocardium but Stoner et a/. (1973) were unable to confirm these results. Studies in dystrophic skeletal muscle (Kar and Pearson, 1972; Neerunjun et al., 1979) have shown increased lysosomal enzyme activities.
mitochondrial
Organelle patholory
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of car~lionryopathy
tance, and to Ms Jean de Luca for typing the manuscript. References Arnold, M. A., Weswig, P. H., Muth, 0.H., and Oldfield, J. E. (1965). Relationship of 5’-nucleotidase activity in lamb muscle to selenium levels administered to their dams. Proceedings of the Society of Experimental Biology and Medicine, 118, 75-76.
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