C A R D I A C BIOPSY I N S K E L E T A L M Y O P A T H Y : R E P O R T OF A C A S E W I T H M Y O C A R D I A L M I T O C H O N D R T A L ABNORMALITIES
R. S . BROWNAND D. PICKERING E. H. MACKAY, Gibson Laboratories, and Departments of Paediatrics and Cardiology, Radclife Znfirniary, Oxford
PLATES XII-XIV T H Eoccurence of various degrees of cardiac involvement in patients with skeletal myopathies has been recognised for nearly a century (Ross, 1883) and indeed, cardiac failure is seen as an important cause of death particularly in the Duchenne type of muscular dystrophy (Perloff, de Leon and O’Doherty, 1966). However, all the published reports of cardiac changes in human skeletal myopathy have been based on post-mortem material examined at the lightmicroscope level only and represent the features of end stage disease. No reIiabIe ultrastructural details are available from these cases so it is perhaps surprising that out of 19 cardiac biopsy series in the literature involving several hundred patients none are recorded as having included patients with overt skeletal myopathy. We therefore wish to report what appears to be the first instance of endomyocardial biopsy in a child with a skeletal myopathy of unusual type and who shows some unique cardiac mitochondria1 abnormalities on electron microscopy. CASEREPORT A. F. was 11 yr old when admitted for investigation of a cardiac murmur first noted at the age of 5 yr, occasional episodes of dizziness without loss of consciousness after strenuous exercise and muscular weakness. His early development was normal but from the age of 4 yr he began to have difficulty in rising from a supine position. He had marked weakness of his neck muscles and occasionally used Gower’s manoeuvre to rise to a standing position. The parents are unrelated and there is no family history of muscular weakness. On examination he was a normally proportioned boy of average height and weight for his age. His pulse rate and blood pressure were normal but there was clinical evidence of biventricular hypertrophy and an ejection systolic murmur at the lower left sternal border. The muscles of the neck, shoulders, upper arms and trunk showed a selective symmetrical weakness and wasting at rest and on testing. All other muscle groups and deep tendon reflexes were normal and there was no fasciculation, myotonia or pseudohypertrophy. His gait was abnormal with an exaggerated lumbar lordosis. His ECG was abnormal with a short PR interval (0.1 s), narrow QRS complexes and abnormal Q and R waves in the chest leads. The chest X-ray confirmed cardiomegaly with a n abnormal globular contour to the heart. Electromyography revealed abnormally brief, polyphasic potentials especially in the left biceps suggesting primary muscle disease. The haemoglobin, ESR, serum electrolytes, creatine kinase, aldolase, pyruvate and lactate plus X-rays of the cervical spine and skull base were all normal. Received 21 July 1975; accepted 31 Oct. 1975. J. PATH.-VOL.
120 (1976)
35
36
E. H. MACKAY, R. S. BROWN AND D. PICKERING
Cardiac catheterisation showed normal right and left-sided pressures with no evidence of an intracardiac shunt and no gradients over the right and left ventricular outflow tracts at rest or after provocation. Cine-angiography outlined a slightly enlarged left ventricle which contracted normally with no asymmetrical hypertrophy or outflow obstruction. A biopsy was taken from the lateral wall of the right ventricle with the child-size Konno endomyocardial bioptome. MATERIALS AND METHODS The cardiac biopsy was divided into two parts for electron microscopy and fixed in icecold 4 per cent. glutaraldehyde, post-fixed in 1 per cent. Osmium tetroxide (both phosphate buffered to pH 7.4), dehydrated through graded ethanol and embedded in Araldite. Thin (0.7 p) sections stained with Azure A were examined by light microscopy and areas selected for further examination. Ultrathin sections of these areas were cut on an LKB Mk 111 Ultratome using glass knives, mounted on uncoated copper grids, stained with aqueous 2 per cent. uranyl acetate and Reynolds lead citrate and examined in a Philips EM 301 electron microscope. A quantitative assessment of the proportionate area occupied by the component parts of each fibre on the electron micrographs was made using a transparent plastic point counting grid based on a system of 1 cm-sided equilateral triangles giving 272 points per photograph.
RESULTS Light microscopy showed some disorderly arrangement of myofibres but no fibrosis and no perinuclear haloes or areas of increased fibre branching. Fibre diameters (measured with an eyepiece micrometer) were in the range 10-30 p with a mean of 18.3 p which is markedly hypertrophied for a child. There was no fibre necrosis, inflammatory cell infiltrate or lipid deposition and small vessels were normal. Electron microscopy revealed a striking and widespread increase in numbers of mitochondria and glycogen granules with a relative reduction of the myofibrillar content of the cardiac muscle cells (fig. 1). The muscle fibres were fully contracted with an average sarcomere length of 1.25 p but no artefactual hypercontraction bands. There was some disorder of fibre orientation and within individual fibres there were areas of fibrillar crossover and branching at angles of up to 90" from the longitudinal axis of the cell. In these areas the Z-bands frequently showed a variable and irregular increase in thickness but the other bands and myofilaments were normally formed and arranged. Elsewhere, the myofibrils were regular, parallel and of essentially normal thickness and arrangement. The sarcolemma showed moderate numbers of pinocytic vesicles and there was some diffuse dilatation of the sarcoplasmic reticulum and the " T " tubules. Scanty, small lipid droplets were present, lysosomes were scarce and only occasional lipofuscin granules were found. Few intercalated discs were seen and these appeared unusually tortuous with prominent fasciae adherens and long side-to-sidejunctions. The nuclei were central and of normal size with minimal peripheral chromatin clumping but no nucleoli or inclusions. Glycogen was present in the p-monoparticulate form and showed a significant increase both generally and as localised collections associated with masses of mitochondria. The most impressive abnormalities were seen in the mitochondria which were greatly increased in number and often in size with abnormal configuration of the cristae and formation of unique ring-shaped forms (figs. 2, 3). By means
37
CARDIAC BIOPSY
of the point counting system the mitochondria in this case were found to occupy between 34.4 and 47.5 per cent. of the area of the fibres examined (see table) compared with the 25 per cent. or so found in normal fibres. The great majority of the mitochondria were more or less circular in outline and presumably spherical in shape with a matrix of average electron density containing distinctly more membrane bound granules of a size and density similar to single glycogen particles than have been seen in other biopsies. Relatively few had a normal pattern of more or less parallel transverse cristae and many had a mosaic array of thin irregular folds while others had a bizarre pattern of tubular cristae criss-crossing or apparently radiating from the centre of the organelle. These mitochondria occupied the usual interfibrillar spaces but with up to five mitochondria per sarcomere. There were no perinuclear or subsarcolemmal collections but many fibres showed massive accumulation of TABLE Quantitative comparison between myocardial cell structure of this patient and essentially normal controls. Results of point counting five fields from each with 272 points per field expressed as percentage of the fibre area in each field This case Component Fibrils Mitochondria Tubules Glycogen Lipofuscin, etc.
Normals
&
&
224-38.8 34.4473 4.1-1 1.7 7'4-28.7 0- 1.2
54.1 246 6.5 10.4 1.4
Range
Mean 33.4 42.8 6.8 16.5 0.4
Mean
Range
44.5-64.5 224-29.7 4.7- 7.9 5.3-14.3 0- 5.7
mitochondria and glycogen particles in their centres often with complete displacement of the myofibrils from these areas (fig. 2). In these foci many of the mitochondria had the spherical shape seen elsewhere but in addition large numbers were long and narrow averaging 2.0x0.2 p in size with irregular transverse cristae. A transition could be seen from the straight rods through curved forms to the ring mitochondria with radial cristae surrounding a glycogen core. One double ring form was present as illustrated (fig. 2) and a double membrane zone indicating a possible fusion site of the meeting ends of the mitochondria could also be seen in several of the rings. The central glycogen core in these ring forms was clearly not within the mitochondria1 lumen or intracristal space as it was surrounded by a well defined double membrane forming the outer wall of the mitochondrion. Histological, histochemical and electron microscopical examination of a skeletal muscle biopsy from the right biceps muscle was entirely normal. DISCUSSION Muscular dystrophy in childhood usually presents in one of three formspseudohypertrophic (Duchenne), limb girdle (Erb), or facio-scapular-humeral (Landouzy-Dejerine). The former is usually present in early childhood while the latter two become manifest in later childhood and early adolescence. The reported incidence of cardiac involvement in children with muscular dystrophy
38
E. H. MACKA Y, R . S. BROWN AND D. PICKERING
had ranged from 25 to 85 per cent. (Perloff et al., 1966), the most usual association being with the pseudohypertrophic form. In the limb girdle form cardiac involvement is rare and non-specific (Walton and Gardner-Medwin, 1969) though electrocardiographic abnormalities are not unusual. However, few of the papers describing the cardiac histopathology in these patients have clearly defined the clinical types of skeletal myopathy present and none have described electron-microscopicappearances, so it is difficult to correlate different forms of cardiac involvement with specific types of muscular dystrophy. To our knowledge there have been no reports of such bizarre mitochondrial changes in cardiomyopathy associated with muscular dystrophy though Kay et al. (1972) reported excess mitochondria in the subsarcolemmal cytoplasm in a case of familial heart block with peroneal muscular atrophy. Our case presents some difficulty in classification, not clinically corresponding with any of the usual forms of muscular dystrophy, but most closely approximating the limb girdle or scapulo-humeral form of Erb (Adams, Denny-Brown and Pearson, 1967). The very striking neck muscle involvement and the other features prompted us to call this proximal muscle dystrophy. Two reports have noted striking weakness of the neck muscles in three patients with so-called mitochondrial myopathies (D’Agostino et al., 1968, Bradley et al., 1969) and one of these illustrates stacking of the cristae in large mitochondria with one possible ring form found in a muscle spindle (D’Agostino et al.). These patients showed no clinical evidence of heart disease and in two the ECG is reported as being normal. Nevertheless, it is possible that neck weakness and mitochondrial myopathy of cardiac and skeletal muscle may represent a previously undescribed combination. There have also been several reports (reviewed by Zacks, 1970) of isolated cases showing major mitochondrial abnormalities in skeletal muscle usually with formation of paracrystalline material in the cristae. Some of these patients had evidence of abnormal enzyme activity in their muscle mitochondria leading to a hypermetabolic state (Luft et al., 1962) or intracellular lipid accumulation (Bradley et al., 1969). Unfortunately the small size of the cardiac muscle biopsy compared with skeletal muscle samples has so far imposed serious limitations on the extent of similar biochemical investigations in cardiomyopathies. Electrocardiographic abnormalities have been extensively described (Skyring and McKusick, 1961, Perloff et al.) and may be the only sign of cardiac involvement in muscular dystrophy. Overall between 30 and 80 per cent. of patients with muscular dystrophy have ECG abnormalities (Gilroy et al., 1963, Welsh, Lynn and Haase, 1963) and in our case the short PR interval, deep Q and tall R waves would be in keeping with these reported changes. Despite the often striking ECG changes and clinical features suggesting heart involvement, cardiac catheterisation, as in our case, rarely reveals any abnormalities (Gailani, Danowski and Fisher, 1958). Interestingly, electromyographic abnormalities of myopathic type have been reported in 65 per cent. of one series of patients with proven hypertrophic obstructive cardiomyopathy (HOCM) and a few also had raised serum creatine phosphokinase and aldolase levels (Meerschwam and Hootsmans, 1971). None
CARDIAC BIOPSY
39
had overt skeletal myopathy but muscular dystrophy was noted in close relatives of two patients. There is thus increasing evidence that some patients with cardiomyopathy may have previously unsuspected skeletal muscle disease as well as vice versa and at least one report describes the onset of cardiomyopathy up to 5 yr before the development of proximal limb girdle muscular dystrophy (Norris, Moss and Yu, 1966). Some of the appearances described in this biopsy are similar to those seen in hypertrophic cardiomyopathy but in that condition there is a greater degree of fibre hypertrophy and branching with perinuclear haloes (Van Noorden, Olsen and Pearse, 1971) and the structural mitochondrial abnormalities seen in our case have not been described. While fibrillar disarray is the principal subcellular feature of HOCM it is seen only rarely in congestive cardiomyopathy but may occasionally be found in simple hypertrophy and as a possible regenerative process at the margin of healing infarcts. Fibril disarray is commonly present in embryonic hearts kept beating in organ culture where the normal orientating forces are not active and is also seen in the atrioventricular node and bundle of His (Ferrans, Morrow and Roberts, 1972). Thus, in our patient this change may represent a genuine dystrophic process affecting the myocardial fibres and be responsible for his rhythm disturbances. The reason for the very marked increase in numbers and size of mitochondria in this patient's myocardium is not easily explained but they are known to contain DNA and can' replicate by simple division. Tandler et a!. (1969) working with riboflavin-deficient mice showed an increase in size of hepatic mitochondria during the stage of deficiency apparently due to failure of division of the enlarging organelles. Budding, division and the frequent formation of ring forms were seen within a few hours of treatment with intraperitoneal riboflavin suggesting that this may be an essential rate limiting factor in mitochondrial replication. It is thus conceivable that a block in riboflavin uptake or utilisation in this patient may to some extent be responsible for the abnormal mitochondrial structures and possibly functions. An alternative explanation for the ring forms-that they are cup-shaped mitochondria with a ring shape determined by the plane of section, seems unlikely in view of the radial arrangement of the cristae and the absence of forms representing other planes of section. The circumference of these ring forms measured around the middle part of the ring is in the range 1.70-3-29 p which is also in keeping with an origin from elongated cylindrical structures. Measurement of mitochondrial diameters on photographs suggests a possible double population of these structures. One is of normal or slightly increased size with diameters of 0.3-0.7 p and the other of 1.14-1.50 p with occasional giant forms up to 2-7 p diameter and relatively few in between. The smaller forms have very disorderly cristae while the larger ones tend to have very regular, closely stacked, parallel cristae (fig. 4) but neither show intramitochondrial crystalloid formation. Assuming the shape to be spherical the larger mitochondria with a diameter of say 1-25 p have a calculated volume of 1.03 p3 which is nearly 10 times the volume of normal mitochondria (0.11 1.3) with diameters of 0.6 p . Thus the
40
E. H . MACKA Y, R. S. BROWN AND D . PICKERINC
increased numbers and volume of these mitochondria offer a greatly increased metabolic potential in the cardiac muscle cells of this patient. No convincing dividing mitochondria were seen in this biopsy so that the size variation may represent a permanent genetically determined abnormality rather than a response to an acquired condition. Turning to the assessment of increased numbers of mitochondria seen on electron microscopy it is apparent that previous estimates of mitochondrial populations have been largely subjective as there are few, if any, published reports of quantitative examination of the ultrastructure of human cardiac muscle. This is partly due to the small size of cardiac biopsies with the attendant possible sampling errors and also to the distortion of muscle cells produced by immersion fixation. However, other reports have also used point counting grids successfully to quantify increases in mitochondria etc. in hypertrophied rat hearts (Page and McCallister, 1973) and Herbener (1973) has shown that, for the rat heart at least, morphometric counting of five fields from one block of right ventricle gives an acceptable level of accuracy. In the present report control material was selected from other patients whose biopsies showed no abnormality on light or electron microscopy and whose sarcomeres showed the same degree ofcontraction, as these are the nearest approach to normal muscle available. Although the mitochondrial proportions found are higher than would be seen in relaxed or semi-contracted muscle the method of counting and control selection enables these figures to be used for comparative purposes with confidence. If this patient’s cardiac muscle abnormality was to be classified on the basis of previously described cases of skeletal myopathy the most likely designation would be the pleoconial myopathy of Shy, Gonatas and Perez (1966). Their patient, an 8-yr-old boy with delayed muscular development, episodic weakness, thirst and a craving for salt, showed a marked increase in numbers and size of mitochondria with stacking and concentric arrangement of cristae in 20-40 per cent. of skeletal muscle fibres. No ring forms were seen and the cardiac status was not recorded. Alternatively, following their precedent, a new term TOROCONIAL myopathy (Latin TORUS-a ring) is suggested to describe the ring-shaped mitochondria which appear to be unique to this particular form of cardiomyopathy. This patient was biopsied as part of a larger series of patients undergoing cardiac biopsy to assess the clinical and pathological diagnostic value of the endomyocardial bioptome in adults and children. So far three children have shown distinct pathological abnormalities making a new diagnosis as in this case and confirming clinical suspicions of endocardial fibroelastosis and hypertrophic cardiomyopathy in the other two. In two more patients the presence of normal endocardium on right ventricular biopsy has reduced a clinical suspicion of endocardial fibroelastosis and biopsy has thus been of value in the investigation of these children. There has been no mortality and no detectable morbidity from this procedure in our hands. While not advocating cardiac biopsy as a routine investigation it is clearly of diagnostic value in selected cases of cardiomyopathy in infants and children.
MACKAY,BROWNA N D PICKERING
PLATEXI1
CARDIAC BIOPSY
FIG. 1 .-Electron micrograph of cardiac biopsy showing myofibres nearly in transverse section. Mitochondria are greatly increased (42.2 per cent. of the area), myofibrils are reduced (34.8 per cent.) and the glycogen content is normal (10.9 per cent.) in this field. The larger mitochondria (arrowed) measure 2.7 p diameter. x 3300.
MACKAY,
PLATEXI11
BROWNA N D PICKERINC
CARDIAC BIOPSY
FIG. 2.-A cardiac muscle fibre showing displacement of myofibrils from the central part and replacement by glycogen (24.8 per cent.) and abnormal mitochondria (47.5 per cent. of area). A possible transition from rod-shaped to ring forms is seen (lettered a-d), plus a double-ring form. x 19.800.
PLATEXIV
MACKAY,BROWNAND PICKERING CARDIAC BIOPSY
FIG.3.-Longitudinal section of a different cardiac myofibre with increased glycogen content (28.7 per cent. of area) and abnormal ring-shaped mitochondria. One mitochondrion has a radiating pattern of tubular cristae (m) and many contain small electron dense granules. There is some fibrillar disorder and irregularity of the Z-bands. x 19,900.
FIG.4.-Enlarged mitochondria 1.3-1.5 p in diameter with closely packed regular cristae surrounded by a mass of monoparticulate glycogen. Smaller forms (0.6-0.9p) show grossly disorganised cristae. x 33,000.
CARDIAC BIOPSY
41
SUMMARY Transvenous right ventricular endomyocardial biopsy in an 1 1-yr-old boy with a proximal skeletal myopathy and a cardiomyopathy, has shown a major increase in mitochondrial size and numbers on electron-microscopic morphometry, with formation of unique ring-shaped mitochondria in cardiac muscle cells, The significance of this finding and the use of cardiac biopsy in children are discussed. We should like to thank Professor P. Sleight who supplied the bioptome and Dr W. A. Littler who took the cardiac biopsy. Mr D. Jerrome produced the electron micrographs. Dr E. H. MacKay is in receipt of a N.H.S. locally organised clinical research grant. REFERENCES ADAMS,R. D., DENNY-BROWN, D., AND PEARSON, C. M. 1967. Diseases of muscle, 2nd ed., Harper and Rowe, New York, p. 324. w. G., HUDGSON,P., GARDNER-MEDWIN, D., AND WALTON, J. N. 1969. Myopathy BRADLEY, associated with abnormal lipid metabolism in skeletal muscle. Lancet, i, 495. M. A., AND BRAY,P. F. 1968. Familial myoD’AGOSTINO, A. N., ZITER,F. A., RALLISON, pathy with abnormal muscle mitochondria. Arch. Neurol., 18, 388. FERRANS, V. J., MORROW, A. G., AND ROBERTS, W. C. 1972. Myocardial ultrastructure in hypertrophic subaortic stenosis. Circulation, 65, 769. GAILANI, S., DANOWSKI, T. S., AND FISHER, D. S. 1958. Muscular dystrophy. Catheterization studies indicating latent congestive heart failure. Circulation, 17, 583. J. L., BERMAN, R., AND NEWMAN, M. 1963. Cardiac and pulmonary GILROY,J., CAHALAN, complications of Duchenne’s progressive muscular dystrophy. Circulation, 27, 484. HERBENER, G. H. 1973. Morphometric comparison of mitochondrial population of normal and hypertrophic hearts. Lab. Invest., 28, 96. w. A., AND MEADE,J. B. 1972. Ulstrastructure of the myocardium in KAY,J. M., LITTLER, familial heart block and peroneal muscular atrophy. Br. Heart J., 34, 1081. LUFT,R., IKKOSS, D., PALMIERI, G., ERNSTER, L., AND AFZELIUS, B. 1962. A case of severe hypermetabolism of non-thyroid origin with a defect in the maintenance of mitochondrial respiratory control : a correlated clinical, biochemical and morphological study. J. Clin. Invest., 41, 1776. MEERSCHWAM, I. S., AND HOOTSMANS, W. J. M. 1971. An electromyographic study in hypertrophic obstructive cardiomyopathy. In Obstructive cardiomyopathy, Ciba Symposium 37, p. 55. NORRIS,F. H., Moss, A. J., AND Yu, P. N. 1966. On the possibility that a type of human muscular dystrophy commences in the myocardium. Ann. N. Y . Acad. Sci., 138, 342, L. P. 1973. Quantitative electron microscopic description of PAGE,E., AND MCCALLISTER, heart muscle cells. Am. J. Cardiol., 31, 172. D. 1966. The cardiomyopathy of proPERLOFF, J. K., DE LEON,A. C., AND O’DOHERTY, gressive muscle dystrophy. Circulation,33, 625. Ross, J. 1883. On a case of pseudohypertrophic paralysis. Br. Med. J., 1, 200. SHY, G. M., GONATAS, N. K., AND PEREZ, M. 1966. Two childhood myopathies with abnormal mitochondria. I. Megaconial myopathy, 11. Pleoconial myopathy. Bruin. 89, 133. SKYRING,A., AND MCKUSICK,V. A. 1961. Clinical, genetic and electrocardiographic studies in childhood muscular dystrophy. Am. J. med. Sci., 242, 534. TANDLER, B., ERLANDSON, R. A., SMITH,A. L., AND WYNDER, E. L. 1969. Riboflavin and mouse hepatic cell structure and function. 11. Division of mitochondria during recovery from simple deficiency. J. Cell Biol., 41, 477.
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E. H. MACKAY, R. S. BROWN AND D. PICKERING
VANNOORDEN, S., OLSEN,E. G. J., AND PEARSE, A. G. E. 1971. Hypertrophic obstructive cardiomyopathy, a histological, histochemical and ultrastructural study of biopsy material. Curdiovusc. Res., 5, 118. WALTON, J. N., AND GARDNER-MEDWIN, D. 1969. In Disorders of voluntary muscle, edited by J. N. Walton, London, Churchill, p. 471. WELSH,J. D., LYNN,T. N., AND HAASE,G. R. 1963. Cardiac findings in 73 patients with muscle dystrophy. Arch. Int. Med., 112, 199. ZACKS,S. I. 1970. Recent contributionsto the diagnosis of muscle disease. Human Path., 1, 465. (140 Refs.)
R. S . BROWNAND D. PICKERING E. H. MACKAY, Gibson Laboratories, and Departments of Paediatrics and Cardiology, Radclife Znfirniary, Oxford
PLATES XII-XIV T H Eoccurence of various degrees of cardiac involvement in patients with skeletal myopathies has been recognised for nearly a century (Ross, 1883) and indeed, cardiac failure is seen as an important cause of death particularly in the Duchenne type of muscular dystrophy (Perloff, de Leon and O’Doherty, 1966). However, all the published reports of cardiac changes in human skeletal myopathy have been based on post-mortem material examined at the lightmicroscope level only and represent the features of end stage disease. No reIiabIe ultrastructural details are available from these cases so it is perhaps surprising that out of 19 cardiac biopsy series in the literature involving several hundred patients none are recorded as having included patients with overt skeletal myopathy. We therefore wish to report what appears to be the first instance of endomyocardial biopsy in a child with a skeletal myopathy of unusual type and who shows some unique cardiac mitochondria1 abnormalities on electron microscopy. CASEREPORT A. F. was 11 yr old when admitted for investigation of a cardiac murmur first noted at the age of 5 yr, occasional episodes of dizziness without loss of consciousness after strenuous exercise and muscular weakness. His early development was normal but from the age of 4 yr he began to have difficulty in rising from a supine position. He had marked weakness of his neck muscles and occasionally used Gower’s manoeuvre to rise to a standing position. The parents are unrelated and there is no family history of muscular weakness. On examination he was a normally proportioned boy of average height and weight for his age. His pulse rate and blood pressure were normal but there was clinical evidence of biventricular hypertrophy and an ejection systolic murmur at the lower left sternal border. The muscles of the neck, shoulders, upper arms and trunk showed a selective symmetrical weakness and wasting at rest and on testing. All other muscle groups and deep tendon reflexes were normal and there was no fasciculation, myotonia or pseudohypertrophy. His gait was abnormal with an exaggerated lumbar lordosis. His ECG was abnormal with a short PR interval (0.1 s), narrow QRS complexes and abnormal Q and R waves in the chest leads. The chest X-ray confirmed cardiomegaly with a n abnormal globular contour to the heart. Electromyography revealed abnormally brief, polyphasic potentials especially in the left biceps suggesting primary muscle disease. The haemoglobin, ESR, serum electrolytes, creatine kinase, aldolase, pyruvate and lactate plus X-rays of the cervical spine and skull base were all normal. Received 21 July 1975; accepted 31 Oct. 1975. J. PATH.-VOL.
120 (1976)
35
36
E. H. MACKAY, R. S. BROWN AND D. PICKERING
Cardiac catheterisation showed normal right and left-sided pressures with no evidence of an intracardiac shunt and no gradients over the right and left ventricular outflow tracts at rest or after provocation. Cine-angiography outlined a slightly enlarged left ventricle which contracted normally with no asymmetrical hypertrophy or outflow obstruction. A biopsy was taken from the lateral wall of the right ventricle with the child-size Konno endomyocardial bioptome. MATERIALS AND METHODS The cardiac biopsy was divided into two parts for electron microscopy and fixed in icecold 4 per cent. glutaraldehyde, post-fixed in 1 per cent. Osmium tetroxide (both phosphate buffered to pH 7.4), dehydrated through graded ethanol and embedded in Araldite. Thin (0.7 p) sections stained with Azure A were examined by light microscopy and areas selected for further examination. Ultrathin sections of these areas were cut on an LKB Mk 111 Ultratome using glass knives, mounted on uncoated copper grids, stained with aqueous 2 per cent. uranyl acetate and Reynolds lead citrate and examined in a Philips EM 301 electron microscope. A quantitative assessment of the proportionate area occupied by the component parts of each fibre on the electron micrographs was made using a transparent plastic point counting grid based on a system of 1 cm-sided equilateral triangles giving 272 points per photograph.
RESULTS Light microscopy showed some disorderly arrangement of myofibres but no fibrosis and no perinuclear haloes or areas of increased fibre branching. Fibre diameters (measured with an eyepiece micrometer) were in the range 10-30 p with a mean of 18.3 p which is markedly hypertrophied for a child. There was no fibre necrosis, inflammatory cell infiltrate or lipid deposition and small vessels were normal. Electron microscopy revealed a striking and widespread increase in numbers of mitochondria and glycogen granules with a relative reduction of the myofibrillar content of the cardiac muscle cells (fig. 1). The muscle fibres were fully contracted with an average sarcomere length of 1.25 p but no artefactual hypercontraction bands. There was some disorder of fibre orientation and within individual fibres there were areas of fibrillar crossover and branching at angles of up to 90" from the longitudinal axis of the cell. In these areas the Z-bands frequently showed a variable and irregular increase in thickness but the other bands and myofilaments were normally formed and arranged. Elsewhere, the myofibrils were regular, parallel and of essentially normal thickness and arrangement. The sarcolemma showed moderate numbers of pinocytic vesicles and there was some diffuse dilatation of the sarcoplasmic reticulum and the " T " tubules. Scanty, small lipid droplets were present, lysosomes were scarce and only occasional lipofuscin granules were found. Few intercalated discs were seen and these appeared unusually tortuous with prominent fasciae adherens and long side-to-sidejunctions. The nuclei were central and of normal size with minimal peripheral chromatin clumping but no nucleoli or inclusions. Glycogen was present in the p-monoparticulate form and showed a significant increase both generally and as localised collections associated with masses of mitochondria. The most impressive abnormalities were seen in the mitochondria which were greatly increased in number and often in size with abnormal configuration of the cristae and formation of unique ring-shaped forms (figs. 2, 3). By means
37
CARDIAC BIOPSY
of the point counting system the mitochondria in this case were found to occupy between 34.4 and 47.5 per cent. of the area of the fibres examined (see table) compared with the 25 per cent. or so found in normal fibres. The great majority of the mitochondria were more or less circular in outline and presumably spherical in shape with a matrix of average electron density containing distinctly more membrane bound granules of a size and density similar to single glycogen particles than have been seen in other biopsies. Relatively few had a normal pattern of more or less parallel transverse cristae and many had a mosaic array of thin irregular folds while others had a bizarre pattern of tubular cristae criss-crossing or apparently radiating from the centre of the organelle. These mitochondria occupied the usual interfibrillar spaces but with up to five mitochondria per sarcomere. There were no perinuclear or subsarcolemmal collections but many fibres showed massive accumulation of TABLE Quantitative comparison between myocardial cell structure of this patient and essentially normal controls. Results of point counting five fields from each with 272 points per field expressed as percentage of the fibre area in each field This case Component Fibrils Mitochondria Tubules Glycogen Lipofuscin, etc.
Normals
&
&
224-38.8 34.4473 4.1-1 1.7 7'4-28.7 0- 1.2
54.1 246 6.5 10.4 1.4
Range
Mean 33.4 42.8 6.8 16.5 0.4
Mean
Range
44.5-64.5 224-29.7 4.7- 7.9 5.3-14.3 0- 5.7
mitochondria and glycogen particles in their centres often with complete displacement of the myofibrils from these areas (fig. 2). In these foci many of the mitochondria had the spherical shape seen elsewhere but in addition large numbers were long and narrow averaging 2.0x0.2 p in size with irregular transverse cristae. A transition could be seen from the straight rods through curved forms to the ring mitochondria with radial cristae surrounding a glycogen core. One double ring form was present as illustrated (fig. 2) and a double membrane zone indicating a possible fusion site of the meeting ends of the mitochondria could also be seen in several of the rings. The central glycogen core in these ring forms was clearly not within the mitochondria1 lumen or intracristal space as it was surrounded by a well defined double membrane forming the outer wall of the mitochondrion. Histological, histochemical and electron microscopical examination of a skeletal muscle biopsy from the right biceps muscle was entirely normal. DISCUSSION Muscular dystrophy in childhood usually presents in one of three formspseudohypertrophic (Duchenne), limb girdle (Erb), or facio-scapular-humeral (Landouzy-Dejerine). The former is usually present in early childhood while the latter two become manifest in later childhood and early adolescence. The reported incidence of cardiac involvement in children with muscular dystrophy
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E. H. MACKA Y, R . S. BROWN AND D. PICKERING
had ranged from 25 to 85 per cent. (Perloff et al., 1966), the most usual association being with the pseudohypertrophic form. In the limb girdle form cardiac involvement is rare and non-specific (Walton and Gardner-Medwin, 1969) though electrocardiographic abnormalities are not unusual. However, few of the papers describing the cardiac histopathology in these patients have clearly defined the clinical types of skeletal myopathy present and none have described electron-microscopicappearances, so it is difficult to correlate different forms of cardiac involvement with specific types of muscular dystrophy. To our knowledge there have been no reports of such bizarre mitochondrial changes in cardiomyopathy associated with muscular dystrophy though Kay et al. (1972) reported excess mitochondria in the subsarcolemmal cytoplasm in a case of familial heart block with peroneal muscular atrophy. Our case presents some difficulty in classification, not clinically corresponding with any of the usual forms of muscular dystrophy, but most closely approximating the limb girdle or scapulo-humeral form of Erb (Adams, Denny-Brown and Pearson, 1967). The very striking neck muscle involvement and the other features prompted us to call this proximal muscle dystrophy. Two reports have noted striking weakness of the neck muscles in three patients with so-called mitochondrial myopathies (D’Agostino et al., 1968, Bradley et al., 1969) and one of these illustrates stacking of the cristae in large mitochondria with one possible ring form found in a muscle spindle (D’Agostino et al.). These patients showed no clinical evidence of heart disease and in two the ECG is reported as being normal. Nevertheless, it is possible that neck weakness and mitochondrial myopathy of cardiac and skeletal muscle may represent a previously undescribed combination. There have also been several reports (reviewed by Zacks, 1970) of isolated cases showing major mitochondrial abnormalities in skeletal muscle usually with formation of paracrystalline material in the cristae. Some of these patients had evidence of abnormal enzyme activity in their muscle mitochondria leading to a hypermetabolic state (Luft et al., 1962) or intracellular lipid accumulation (Bradley et al., 1969). Unfortunately the small size of the cardiac muscle biopsy compared with skeletal muscle samples has so far imposed serious limitations on the extent of similar biochemical investigations in cardiomyopathies. Electrocardiographic abnormalities have been extensively described (Skyring and McKusick, 1961, Perloff et al.) and may be the only sign of cardiac involvement in muscular dystrophy. Overall between 30 and 80 per cent. of patients with muscular dystrophy have ECG abnormalities (Gilroy et al., 1963, Welsh, Lynn and Haase, 1963) and in our case the short PR interval, deep Q and tall R waves would be in keeping with these reported changes. Despite the often striking ECG changes and clinical features suggesting heart involvement, cardiac catheterisation, as in our case, rarely reveals any abnormalities (Gailani, Danowski and Fisher, 1958). Interestingly, electromyographic abnormalities of myopathic type have been reported in 65 per cent. of one series of patients with proven hypertrophic obstructive cardiomyopathy (HOCM) and a few also had raised serum creatine phosphokinase and aldolase levels (Meerschwam and Hootsmans, 1971). None
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had overt skeletal myopathy but muscular dystrophy was noted in close relatives of two patients. There is thus increasing evidence that some patients with cardiomyopathy may have previously unsuspected skeletal muscle disease as well as vice versa and at least one report describes the onset of cardiomyopathy up to 5 yr before the development of proximal limb girdle muscular dystrophy (Norris, Moss and Yu, 1966). Some of the appearances described in this biopsy are similar to those seen in hypertrophic cardiomyopathy but in that condition there is a greater degree of fibre hypertrophy and branching with perinuclear haloes (Van Noorden, Olsen and Pearse, 1971) and the structural mitochondrial abnormalities seen in our case have not been described. While fibrillar disarray is the principal subcellular feature of HOCM it is seen only rarely in congestive cardiomyopathy but may occasionally be found in simple hypertrophy and as a possible regenerative process at the margin of healing infarcts. Fibril disarray is commonly present in embryonic hearts kept beating in organ culture where the normal orientating forces are not active and is also seen in the atrioventricular node and bundle of His (Ferrans, Morrow and Roberts, 1972). Thus, in our patient this change may represent a genuine dystrophic process affecting the myocardial fibres and be responsible for his rhythm disturbances. The reason for the very marked increase in numbers and size of mitochondria in this patient's myocardium is not easily explained but they are known to contain DNA and can' replicate by simple division. Tandler et a!. (1969) working with riboflavin-deficient mice showed an increase in size of hepatic mitochondria during the stage of deficiency apparently due to failure of division of the enlarging organelles. Budding, division and the frequent formation of ring forms were seen within a few hours of treatment with intraperitoneal riboflavin suggesting that this may be an essential rate limiting factor in mitochondrial replication. It is thus conceivable that a block in riboflavin uptake or utilisation in this patient may to some extent be responsible for the abnormal mitochondrial structures and possibly functions. An alternative explanation for the ring forms-that they are cup-shaped mitochondria with a ring shape determined by the plane of section, seems unlikely in view of the radial arrangement of the cristae and the absence of forms representing other planes of section. The circumference of these ring forms measured around the middle part of the ring is in the range 1.70-3-29 p which is also in keeping with an origin from elongated cylindrical structures. Measurement of mitochondrial diameters on photographs suggests a possible double population of these structures. One is of normal or slightly increased size with diameters of 0.3-0.7 p and the other of 1.14-1.50 p with occasional giant forms up to 2-7 p diameter and relatively few in between. The smaller forms have very disorderly cristae while the larger ones tend to have very regular, closely stacked, parallel cristae (fig. 4) but neither show intramitochondrial crystalloid formation. Assuming the shape to be spherical the larger mitochondria with a diameter of say 1-25 p have a calculated volume of 1.03 p3 which is nearly 10 times the volume of normal mitochondria (0.11 1.3) with diameters of 0.6 p . Thus the
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E. H . MACKA Y, R. S. BROWN AND D . PICKERINC
increased numbers and volume of these mitochondria offer a greatly increased metabolic potential in the cardiac muscle cells of this patient. No convincing dividing mitochondria were seen in this biopsy so that the size variation may represent a permanent genetically determined abnormality rather than a response to an acquired condition. Turning to the assessment of increased numbers of mitochondria seen on electron microscopy it is apparent that previous estimates of mitochondrial populations have been largely subjective as there are few, if any, published reports of quantitative examination of the ultrastructure of human cardiac muscle. This is partly due to the small size of cardiac biopsies with the attendant possible sampling errors and also to the distortion of muscle cells produced by immersion fixation. However, other reports have also used point counting grids successfully to quantify increases in mitochondria etc. in hypertrophied rat hearts (Page and McCallister, 1973) and Herbener (1973) has shown that, for the rat heart at least, morphometric counting of five fields from one block of right ventricle gives an acceptable level of accuracy. In the present report control material was selected from other patients whose biopsies showed no abnormality on light or electron microscopy and whose sarcomeres showed the same degree ofcontraction, as these are the nearest approach to normal muscle available. Although the mitochondrial proportions found are higher than would be seen in relaxed or semi-contracted muscle the method of counting and control selection enables these figures to be used for comparative purposes with confidence. If this patient’s cardiac muscle abnormality was to be classified on the basis of previously described cases of skeletal myopathy the most likely designation would be the pleoconial myopathy of Shy, Gonatas and Perez (1966). Their patient, an 8-yr-old boy with delayed muscular development, episodic weakness, thirst and a craving for salt, showed a marked increase in numbers and size of mitochondria with stacking and concentric arrangement of cristae in 20-40 per cent. of skeletal muscle fibres. No ring forms were seen and the cardiac status was not recorded. Alternatively, following their precedent, a new term TOROCONIAL myopathy (Latin TORUS-a ring) is suggested to describe the ring-shaped mitochondria which appear to be unique to this particular form of cardiomyopathy. This patient was biopsied as part of a larger series of patients undergoing cardiac biopsy to assess the clinical and pathological diagnostic value of the endomyocardial bioptome in adults and children. So far three children have shown distinct pathological abnormalities making a new diagnosis as in this case and confirming clinical suspicions of endocardial fibroelastosis and hypertrophic cardiomyopathy in the other two. In two more patients the presence of normal endocardium on right ventricular biopsy has reduced a clinical suspicion of endocardial fibroelastosis and biopsy has thus been of value in the investigation of these children. There has been no mortality and no detectable morbidity from this procedure in our hands. While not advocating cardiac biopsy as a routine investigation it is clearly of diagnostic value in selected cases of cardiomyopathy in infants and children.
MACKAY,BROWNA N D PICKERING
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FIG. 1 .-Electron micrograph of cardiac biopsy showing myofibres nearly in transverse section. Mitochondria are greatly increased (42.2 per cent. of the area), myofibrils are reduced (34.8 per cent.) and the glycogen content is normal (10.9 per cent.) in this field. The larger mitochondria (arrowed) measure 2.7 p diameter. x 3300.
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FIG. 2.-A cardiac muscle fibre showing displacement of myofibrils from the central part and replacement by glycogen (24.8 per cent.) and abnormal mitochondria (47.5 per cent. of area). A possible transition from rod-shaped to ring forms is seen (lettered a-d), plus a double-ring form. x 19.800.
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MACKAY,BROWNAND PICKERING CARDIAC BIOPSY
FIG.3.-Longitudinal section of a different cardiac myofibre with increased glycogen content (28.7 per cent. of area) and abnormal ring-shaped mitochondria. One mitochondrion has a radiating pattern of tubular cristae (m) and many contain small electron dense granules. There is some fibrillar disorder and irregularity of the Z-bands. x 19,900.
FIG.4.-Enlarged mitochondria 1.3-1.5 p in diameter with closely packed regular cristae surrounded by a mass of monoparticulate glycogen. Smaller forms (0.6-0.9p) show grossly disorganised cristae. x 33,000.
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SUMMARY Transvenous right ventricular endomyocardial biopsy in an 1 1-yr-old boy with a proximal skeletal myopathy and a cardiomyopathy, has shown a major increase in mitochondrial size and numbers on electron-microscopic morphometry, with formation of unique ring-shaped mitochondria in cardiac muscle cells, The significance of this finding and the use of cardiac biopsy in children are discussed. We should like to thank Professor P. Sleight who supplied the bioptome and Dr W. A. Littler who took the cardiac biopsy. Mr D. Jerrome produced the electron micrographs. Dr E. H. MacKay is in receipt of a N.H.S. locally organised clinical research grant. REFERENCES ADAMS,R. D., DENNY-BROWN, D., AND PEARSON, C. M. 1967. Diseases of muscle, 2nd ed., Harper and Rowe, New York, p. 324. w. G., HUDGSON,P., GARDNER-MEDWIN, D., AND WALTON, J. N. 1969. Myopathy BRADLEY, associated with abnormal lipid metabolism in skeletal muscle. Lancet, i, 495. M. A., AND BRAY,P. F. 1968. Familial myoD’AGOSTINO, A. N., ZITER,F. A., RALLISON, pathy with abnormal muscle mitochondria. Arch. Neurol., 18, 388. FERRANS, V. J., MORROW, A. G., AND ROBERTS, W. C. 1972. Myocardial ultrastructure in hypertrophic subaortic stenosis. Circulation, 65, 769. GAILANI, S., DANOWSKI, T. S., AND FISHER, D. S. 1958. Muscular dystrophy. Catheterization studies indicating latent congestive heart failure. Circulation, 17, 583. J. L., BERMAN, R., AND NEWMAN, M. 1963. Cardiac and pulmonary GILROY,J., CAHALAN, complications of Duchenne’s progressive muscular dystrophy. Circulation, 27, 484. HERBENER, G. H. 1973. Morphometric comparison of mitochondrial population of normal and hypertrophic hearts. Lab. Invest., 28, 96. w. A., AND MEADE,J. B. 1972. Ulstrastructure of the myocardium in KAY,J. M., LITTLER, familial heart block and peroneal muscular atrophy. Br. Heart J., 34, 1081. LUFT,R., IKKOSS, D., PALMIERI, G., ERNSTER, L., AND AFZELIUS, B. 1962. A case of severe hypermetabolism of non-thyroid origin with a defect in the maintenance of mitochondrial respiratory control : a correlated clinical, biochemical and morphological study. J. Clin. Invest., 41, 1776. MEERSCHWAM, I. S., AND HOOTSMANS, W. J. M. 1971. An electromyographic study in hypertrophic obstructive cardiomyopathy. In Obstructive cardiomyopathy, Ciba Symposium 37, p. 55. NORRIS,F. H., Moss, A. J., AND Yu, P. N. 1966. On the possibility that a type of human muscular dystrophy commences in the myocardium. Ann. N. Y . Acad. Sci., 138, 342, L. P. 1973. Quantitative electron microscopic description of PAGE,E., AND MCCALLISTER, heart muscle cells. Am. J. Cardiol., 31, 172. D. 1966. The cardiomyopathy of proPERLOFF, J. K., DE LEON,A. C., AND O’DOHERTY, gressive muscle dystrophy. Circulation,33, 625. Ross, J. 1883. On a case of pseudohypertrophic paralysis. Br. Med. J., 1, 200. SHY, G. M., GONATAS, N. K., AND PEREZ, M. 1966. Two childhood myopathies with abnormal mitochondria. I. Megaconial myopathy, 11. Pleoconial myopathy. Bruin. 89, 133. SKYRING,A., AND MCKUSICK,V. A. 1961. Clinical, genetic and electrocardiographic studies in childhood muscular dystrophy. Am. J. med. Sci., 242, 534. TANDLER, B., ERLANDSON, R. A., SMITH,A. L., AND WYNDER, E. L. 1969. Riboflavin and mouse hepatic cell structure and function. 11. Division of mitochondria during recovery from simple deficiency. J. Cell Biol., 41, 477.
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VANNOORDEN, S., OLSEN,E. G. J., AND PEARSE, A. G. E. 1971. Hypertrophic obstructive cardiomyopathy, a histological, histochemical and ultrastructural study of biopsy material. Curdiovusc. Res., 5, 118. WALTON, J. N., AND GARDNER-MEDWIN, D. 1969. In Disorders of voluntary muscle, edited by J. N. Walton, London, Churchill, p. 471. WELSH,J. D., LYNN,T. N., AND HAASE,G. R. 1963. Cardiac findings in 73 patients with muscle dystrophy. Arch. Int. Med., 112, 199. ZACKS,S. I. 1970. Recent contributionsto the diagnosis of muscle disease. Human Path., 1, 465. (140 Refs.)
