Endomyocardial biopsies for early detection of mitochondrial disorders in hypertrophic cardiomyopathies

Endomyocardial biopsies for early detection of mitochondrial disorders in hypertrophic cardiomyopathies Pierre Rustin, PhD, J e r 6 m e Lebidois, MD, D o m i n i q u e C h r e t i e n , PhD, Thomas Bourgeron, PhD, Jean-Frangois P i e c h a u d , MD, A g n 6 s ROtig, PhD, Arnold Munnich, MD, PhD, a n d Daniel Sidi, MD From the D6partement de P&diatrie and Unit6 de Recherches sur les Handicaps Gen6tiques de I'Enfant, INSERMU-fl2, H6pital des Enfants-Malades, Paris, France Considering the high proportion of u n e x p l a i n e d hypertrophic c a r d i o m y o p a thies on the one hand and the o c c u r r e n c e of cardiomyopathies in several mitochondrial disorders on the other, we hypothesized that isolated hypertrophic cardiomyopathies in infancy could o c c a s i o n a l l y be the result of defects of oxidative phosphorylation. By means of a scaled-down technique, we were a b l e to investigate oxidative phosphorylation on minute amounts of e n d o m y o c a r dial tissue (I mg) in three patients with concentric hypertrophic c a r d i o m y o p a t h y (shortening fraction in diameter, 18% to 27%; normal mean _+ 1 SD, 33 _+ 3%) and in control subjects. Although the absolute respiratory chain enzyme activities in the e n d o m y o c a r d i a l biopsy specimens of the patients were within the low normal range, the determination of the activity ratios a l l o w e d us to ascribe hypertrophic c a r d i o m y o p a t h i e s to respiratory chain enzyme abnormalities in all three cases ( c o m p l e x I, two cases; multiple enzyme deficiency, one case). The respiratory chain enzyme activity ratios, which are normally constant irrespective of the tissue tested, were markedly abnormal in all three patients ( c y t o c h r o m e c o x i d a s e / r e d u c e d n i c o t i n a m i d e - a d e n i n e d i n u c l e o t i d e c y t o c h r o m e creductase, 4.6 to 10.4; normal mean _+ I SD, 2.9 +_ 0.5). We c o n c l u d e that mitochondrial disorders should be regarded as potential causes of hypertrophic c a r d i o m y o p a t h y in early infancy. Because c a r d i a c catheterization is routinely performed for hem o d y n a m i c investigation of cardiomyopathies, we suggest that e n d o m y o c a r dial biopsies be considered as a tool for early d e t e c t i o n of mitochondrial cardiomyopathies, especially in hypertrophic forms of the disease. (J PEDIATR1994; 124:224-8) Hypertrophic cardiomyopathies are primary myocardial diseases characterized by hypertrophy of the myocardium with no significant cardiac dilation or inflammation.1 Several genetic diseases are known to cause hypertrophic car-

Supported by Association Franqaise contre les Myopathies and Minist6re de la Recherche et de la Technologie. Submitted for publication Dec. 7, 1992; accepted Sept. 16, 1993. Reprint requests: Arnold Munnich, MD, PhD, D6partement de P6diatrie, INSERM U-12, HSpital des Enfants-Malades, 149 rue de SSvres, 75743 Paris--Cedex 15, France. Copyright ® 1994 by Mosby-Year Book, Inc. 0022-3476/94 $3.00 + 0 9/20/51534

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diomyopathy, including glycogen and lysosomal storage diseases, hemochromatosis, Noonan syndrome, progressive muscular dystrophy, degenerative disorders (tuberous scleBSA COX EMB NCCR SCCR

Bovine serum albumin Cytochrome c oxidase Endomyocardial biopsy Nicotinamide-adenine dinucleotide (reduced) (NADH) cytochrome e reductase Succinate cytochrome c reductase

rosis, Friedreich ataxia) and, more recently, the dominantly inherited mutations in the myosine heavy chain. 2 Never-

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Table I. Clinical and genetic features of three patients with hypertrophic cardiomyopathy Patient 1

Sex Birth weight and gestational age Ethnic origin Family history Age of onset Initial symptoms Echographic findings LVED (d) SF PLVWD (d) IS/PLVWD Clinical outcome

F 3050 gm 36 wk Portuguese Fatal heart failure in a sibling aged 6 wk 3 mo Bronchitis, perioral cyanosis, floppiness

Patient 2

M 2500 gm 39 wk Black African First-cousin parents, miscarriages 10 days Apneas, perioral cyanosis

Patient 3

M 2270 gm 38 wk Tunisian One healthy sister, two affected brothers 10 wk Polypnea, perioral cyanosis

35 (+1.5) 27% 8.5 (+4) 1.05 Stationary

23 (+0.5) 32 (+1.5) 18% 23% 7.8 (+5) 6.6 (+3) 1.0 1.0 Left ventricular failure (3 Terminal ventricular ran) failure (4 ran) LVED, Left ventricularend-diastolicdiameter(in millimeters);d, divergencefrom normal,expressedas a multipleof standarddeviationaccordingto the method of Rog6et al.9;SF, shorteningfractionin diameter(%) (i.e.,end-diastolicminusend-systolic/end-diastolicratio;normal = 33 + 3%);PLVWD, posteriorleft ventricutar wall thicknessin diastole(in millimeters);IS/PLVWD, interventricularseptum posteriorleft free wall thicknessratio (normal, <1.3). theless, most hypertrophic cardiomyopathies remain unexplained, and the question of whether and how they are inherited remains largely unanswered. The impairment of the mitochondrial respiratory chain has been shown to result in a variety of clinical manifestations that sometimes included cardiomyopathy. 38 Because of the high rate of unexplained hypertrophic cardiomyopathies on the one hand and the occasional occurrence of cardiomyopathies in mitochondrial disorders on the other, we decided to investigate unexplained cardiomyopathies prospectively for possible defects of oxidative phosphorylation in infancy. PATIENTS The initial symptoms of cardiomyopathy in our patients included hyperpnea, poor sucking, perioral cyanosis, fatigue, floppiness, and bronchitis in the first few weeks of life, but no clinical evidence of extracardiac involvement was observed (Table I). The patients were born to healthy parents after normal pregnancies and deliveries. First-degree consanguinity, previous cases of cardiomyopathy, or both were noted in the three pedigrees. Echocardiography showed increased left ventricle posterior wall thickness in diastole (>4 SD) and a dilated and hypokinetic left ventricle (increased left ventricular end-diastolic diameter; Table I). No asymmetric ventricular hypertrophy (normal septalleft ventricle posterior wall thickness; see Table I and reference 9), subaortic occlusion, fiber disarray (not shown), or family history of cardiomyopathy in previous generations was noted, precluding dominant forms of the disease. 2 Hypertrophic cardiomyopathies associated with muscular dystrophy or lysosomal storage diseases were also excluded,

Similarly, no chromosomal abnormalities, neutropenia, or associated malformations were observed, which ruled out Noonan syndrome, Barth syndrome, 1° and other syndromic associations as the cause of the cardiomyopathy. METHODS

Metabolic investigations in patients. Blood glucose, lactate, pyruvate, and ketone body levels and their molar ratios were determined after deproteinization by perchloric acid in both fasted and fed individuals. 1l This procedure allows detection of (1) postabsorptive hyperlactatemia and elevated lactate/pyruvate and ketone body molar ratios, related to impaired oxidative phosphorylation, and (2) fasting hypoketotic hypoglycemia related to impaired oxidation of fatty acids in liver. Our patients had no hypoketotic hypoglycemia, low plasma carnitine values, urinary excretion of specific metabolites, or abnormal in vitro oxidation of fatty acids labeled with carbon 14 in circulating lymphocytes, which precluded inborn errors of fatty acid B-oxidation (not shown). Biochemical, histnpathologic, and molecular investigations of endomyocardial biopsy specimens and skeletal muscle. Scaled-down EMB specimens of the right ventricle (1 rag) were obtained from the patients with the use of local anesthesia during cardiac catheterization after informed consent of the parents and with the approval of the medical ethics committee. Procedures for EMB have been previously reported I2 and are widely used for the diagnosis of heart rejection after cardiac transplantation. Tissue samples were immediately placed in sterile polypropylene vials and were freeze dried in liquid nitrogen. Control subjects (eight children with thalassemia, 2 to 16 years of age) un-

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T a b l e II. Respiratory chain enzyme activities in three patients with hypertrophic cardiomyopathy

Activities (nmol/min/mg protein) COX Endomyocardial biopsy Patient 1 Patient 2 Patient 3 Control subjects (n = 8) Valvular stenosis (n = 3) Skeletal muscle homogenate Patient 1 Patient 3 Control subjects (n = 14)

1052 1170 370 105-1301 618-1229 647 24 37-136

Ratios (mean ± SD)

Enzyme defect

SCCR

NCCR

COX/SCCR

COX/NCCR

SCCR/NCCR

303 315 74 33-393 171-394

101 170 80 40-502 184-464

3.5 3.7 5.0 3.4 _+ 0.4 3.4 _+ 0.3

10.4 6.8 4.6 2.9 + 0.5 3.0 _+ 0.4

3.0 1.9 1.2 0.9 _+ 0.2 0.9 _+ 0.1

Complex I Complex I Multiple

215 9 11-37

54 7 8-57

3.0 2.7 3.4 _+ 0.3

12 3.4 3.3 _+ 0.7

4.0 1.3 0.9 _+ 0.2

Complex I Multiple

Skeletal muscle mitochondria Patient 1 6124 1941 453 3.1 14 4.3 Complex I Patient 2 1635 582 196 2.8 8.4 3.0 Complex I Control subjects (n = 11) 194-2655 63-803 36-1288 3.1 _+ 0.5 3.4 _+ 0.7 1.1 _+ 0.2 Activityvalueswere the meansof at least three determinations,and maximalstandarddeviationassociatedwith these measurementswas < 11%. derwent cardiac catheterization for quantification of iron deposits in the myocardium. The control subjects met clinical, hemodynamic, and echographic criteria for normal cardiac function. To investigate the specific effect of abnormal loading conditions, we also studied three patients with severe pulmonary stenosis (right ventricular pressure >200 mm Hg). Homogenates from frozen EMB specimens were prepared in 30:1 volumes of sucrose, 0.25 mol/L, potassium chloride, 40 mmol/L, ethylenediaminetetraacetic acid, 3 mmol/L, bovine serum albumin, 1 mg/ml, and Tris HC120 mmol/L (pH 7.4). Tissues were disrupted in a 0.5 ml glass tube by three strokes of a ground-glass pestle. The homogenate was centrifuged at 2000g for 3 minutes. Enzyme activities were spectrophotometrically measured by following either the oxidation or the reduction of cytochrome c at 550 nm according to previously published procedures. 13 Cytochrome c oxidase activity (EC 1.9.3.1) was measured in phosphate buffer, 10 mmol/L (pH 6.5), BSA, 1 mg/ml, with reduced cytochrome e, 10 #mol/L and n-dodecyl B-Dmaltoside, 1.25 mmol/L, used as detergent. Succinate cytochrome c reductase was assayed in phosphate buffer, 10 mmol/L (pH 7.8), containing BSA, 1 mg/ml, potassium cyanide, 0.2 mmol/L, rotenone, 3 gmol/L, adenosine triphosphate, 0.1 mmol/L, and oxidized cytochrome c, 40 gmol/L. Rotenone-sensitive reduced nicotinamide-adenine dinucleotide cytochrome c reductase was assayed as previously described.14 Mitochondria were first burst by a hypotonic shock in distilled water and the activity was measured in phosphate buffer, 10 mmol/L (pH 8.0), containing BSA, 1 mg/ml, potassium cyanide, 0.2 mmol/L and oxidized cytochrome c, 40 #mol/L. Respiratory enzyme activities in EMB specimens from control subjects did not significantly

differ from those found in surgical or immediate postmortem biopsy specimens of the myocardium. 15 Mitochondria from skeletal muscle biopsies (150 rag, deltoid) were prepared as previously described. 14 Spectrophotometric studies on muscle mitochondria and homogenates were carried out as previously described. Because a balanced proportion between respiratory chain complexes is required for the normal functioning of the respiratory chain, 16 enzyme activities were presented both as absolute values and as ratios. The protein concentration was determined by the method of Bradford 17with BSA as a standard. For light microscopy histochemistry, endomyocardial and skeletal muscle biopsy specimens were stained by standard and histoenzymologic techniques, is For analysis of heart mitochondrial DNA by Southern blotting, total DNA, derived from the pooled residues of the EMB specimen after enzyme studies, was digested, separated by agarose gel (0.7%), electrophorescd, and transferred onto nylon filters (Hybond N+; Amersham International, Little Chalfont, United Kingdom). The filters were next hybridized with a full-length mitochondrial DNA probe labeled with phosphorus 32-deoxycytidine triphosphate. RESULTS High rates of respiratory enzyme activities were found in the EMB specimens of control individuals (Table II). Although absolute values in the myocardium were scattered over almost two orders of magnitude, the normal activity ratios showed a narrow range of values (mean C O X / S C C R ratio +_ 1 SD = 3.4 +_ 0.4; mean S C C R / N C C R ratio _+ 1 SD = 0.9 + 0.2; Table II). A normal range of normal respiratory chain enzyme activity ratios has been observed for

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Table III, Oxidoreduction status in plasma of three patients with hypertrophic cardiomyopathy

Lactate (mmol/L) Patient 1 Fasted 3.70 Fed 7.90 Patient 2 Fasted 4.65 Fed 4.01 Patient 3 Fasted 2.50 Fed 2.31 Control subjects (n = 27) Fasted 0.8-1.5 Fed 0.6-2.4

Pyruvate (mmol/L)

L/P

f l - O H - B u t y r a t e Acetoacetate (mmol/L) (mmol/L)

fl-OHBut/Ac

Bloodglucose (mmol/L)

0.17 0.26

21.8 30.4

0.92 0.69

015 0.14

6.1 4.9

4.6 5.6

0.23 0.20

20.2 20.1

0.13 0.06

0.06 0.06

2.0 1.0

4.7 5.6

0.13 0.10

19.2 23.0

0.025 <0.02

0.08 0.08

0.3 0.3

2.3 6.7

0.05-0.2 0.03-0.2

<20 <20

0.1-0.9 0.02-0.09

0.05-0.1 0.01-0.04

1.2-3.2 <1

3.5-4.8 5-6

Blood sampleswere taken either after a 12-hoarfast (fasted) or 1 hour after a normalmeal (fed). L/P, Lactate/pyruvatemolar ratio; fl-OH-But/Ac:/3-OH-butyrate-acetoaeetatemolar ratio. COX, SCCR, and NCCR irrespective of the human tissue tested.15, 16 Consequently, individuals whose absolute values were in the low normal range could have a diagnosis of enzyme deficiency made, as already shown in liver, muscle, and cultured skin fibroblasts of patients with respiratory chain enzyme deficiencies. I921 In addition, the determination of the activity ratios allowed characterization of the respiratory enzyme activities without reference to the mitochondrial protein content, especially since this content can vary in mitochondrial disorders. Table I! shows that the three patients with hypertrophic cardiomyopathy had abnormal respiratory chain enzyme activity ratios in their EMB specimens, a finding that suggests a defect in complex I (two patients) and a multiple enzyme deficiency (one patient). Lipid droplets were found in their EMB specimens, and concordant results were observed in affected siblings (patient 3, not shown). Ventricle load and heart failure were ruled out as causes of respiratory enzyme deficiency because specific activities were not altered in patients with either dilated cardiomyopathy (not shown) or pulmonary valvular stenosis, despite very high pressure loads in the right ventricle (>200 mm Hg, Table II). Molecular analysis of mitochondrial DNA revealed no major deletion of the mitochondrial genome in any patient (not shown). A similar imbalance of respiratory enzyme activities was found both in skeletal muscle and in specimens of the three patients (Table II). However, in patient 1 this was associated with a high level of respiratory enzyme activities. This feature might be related to the accumulation of defective mitochondria with a low protein content, which would result in an apparent increase of enzyme-specific activities. The three patients had large lipid droplets with subsarcolemmal accumulation of mitochondria in their type I skeletal muscle fibers, and lactate concentrations and/or

lactate/pyruvate ratios were slightly elevated in their plasma (Table III). DISCUSSION We describe the EMB as a tool for the diagnosis of mitochondrial cardiomyopathies. By means of a scaled-down technique, we were able to investigate oxidative phosphorylation on minute amounts of endomyocardial tissue (1 mg) and, on the basis of enzyme activity ratios, to ascribe isolated concentric hypertrophic cardiomyopathy to respiratory enzyme abnormalities in three affected children. A balanced proportion of each respiratory chain complex is required for the normal functioning of the mitochondrial respiratory chain. This feature accounts for the constant ratios of respiratory chain enzyme activities consistently found in all normal tissues, including heart tissue. 15, 16 For these reasons, patients with absolute enzyme activities in EMB specimens that were in the low normal range could have an unambigous diagnosis of enzyme deficiency, as already shown in liver, muscle, and cultured fibroblasts. 1921 Ventricle load, heart failure, or myocardial hypertrophy were ruled out as possible causes of secondary deficiencies for two reasons. First, the enzyme activities were not altered in patients with pulmonary valvular stenosis. Second, several patients with severely hypertrophic or dilated cardiomyopathies had normal enzyme activities in their EMB specimensY Therefore the abnormal activity ratios in our study cannot be regarded as the nonspecific consequences of heart failure or hypertrophy. Although the skeletal muscle of the three patients was not clinically affected, abnormal activity ratios could also be measured in this tissue. This could be related in part to the rapidly fatal course of the heart failure, which precluded a wider clinical expression of the disease. The question of whether heart-specific defects of oxidative phosphorylation

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exist remains open to debate, especially since tissue-specific isoenzymes have been described in the mitochondrial respiratory chain. 23 Screening for respiratory enzyme deficiencies by E M B represents the only chance of recognizing isolated mitochondrial cardiomyopathies, the frequency of which among unexplained cardiomyopathies is unknown. Future studies of hypertrophic, dilated, and restrictive cardiomyopathies should address this important issue. Cardiomyopathies have been shown to occur in the course of mitochondrial disorders with initial extracardiac manifestations. 3"8 Hitherto, however, cardiomyopathy has not been reported as the initial symptom of a mitochondrial disorder. This study suggests that defects of oxidative phosphorylation can be manifested initially as a cardiomyopathy and should now be regarded as a cause of isolated hypertrophic cardiomyopathy in early infancy, especially when an abnormal oxidation-reduction status is found in plasma studies. Consanguinity, family history of cardiomyopathy, or both further support the inherited nature of this condition. Finally, because cardiac catheterization is routinely performed for the hemodynamic investigation of cardiornyopathies, we suggest that E M B be considered a tool for early detection of mitochondrial cardiomyopathies, especially in hypertrophic forms of the disease. REFERENCES

1. Maron BJ. Cardiomyopathies. In: Adams FH, Emmanouilides GC, Reimenschneider TA, eds. Heart disease in infants, children and adolescents. 4th ed. Baltimore, Maryland: Williams & Wilkins, 1992:940-64. 2. Jarcho JA, McKenna WMc, Pare 3AP, et al. Mapping a gene for familial hypertrophic cardiomyopathy to chromosome 14ql. N Engl J Med 1989;321:1372-8. 3. Moreadith RW, Batshaw ML, Ohnishi T, et al. Deficiency of the iron-sulfur clusters of mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase (complex I) in an infant with congenital lactic acidosis. J Clin Invest 1984;74:685-97. 4. Papadimitriou A, Neustein HB, DiMauro S, Stanton R, Bressolin N. Histiocytoid cardiomyopathy of infancy: deficiency of reducible cytochrome b in heart mitochondria. Pediatr Res 1984;18:1023-8. 5. Robinson BH, Ward J, Goodyer P, Beaudet A. Respiratory chain defects in the mitochondria of cultured skin fibroblasts from three patients with lacticacidemia. J Clin Invest 1986; 77:1422-7. 6. DiMauro S, Zeviani M, Bonilla E, et al. Cytochrome c oxidase deficiency. Biochem Soc Trans 1985;13:651-3.

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7. Zeviani M, Gellera G, Antozzi C, et al. Maternally inherited myopathy and cardiomyopathy: association with mutation in mitochondrial DNA tRNA Leu(wUR). Lancet 1991;338:143-7. 8. Wallace DC. Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science 1992;256:628-32. 9. Rog6 C, Silverman NH, Hart PA, Ray RM. Cardiac structure growth pattern determined by echocardiography. Circulation 1978;57:285-90. 10. Bolhuis PA, Hensels GW, Hulsebos TJM, Baas F, Barth PG. Mapping of the locus for X-linked cardioskeletal myopathy with neutropenia and abnormal mitochondria (Barth syndrome) to Xq28. Am J Hum Genet 1991;48:481-5. 11. R6tig A, Cormier V, Blanche S, et al. Pearson's marrow-pancreas syndrome: a multisystem mitochondrial disorder in infancy. J Clin Invest 1990;86:1601-8. 12. Mason JW. Techniques for right and left ventricular endomyocardial biopsy. Am J Cardiol 1978;41:887-92. 13. Rustin P, Chretien D, Bourgeron T, et al. Biochemical and molecular investigation of respiratory chain deficiencies. Clin Chim Acta (In press). 14. Chretien D, Bourgeron T, Rftig A, Munnich A, Rustin P. The measurement of the rotenone-sensitive NADH cytoehrome c reductase activity in mitochondria isolated from minute amount of human skeletal muscle. Biochem Biophys Res Commun 1990;173:26-33. 15. Rustin P, Chretien D, Bourgeron T, et al. Investigation of respiratory chain activity in human heart. Biochem Med Metab Biol 1993;50:120-6. 16. Rustin P, Chretien D, Bourgeron T. Assessment of the mitochondrial respiratory chain. Lancet 1991;338:60. 17. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 1976;72:248-54. 18. Dubowitz V, Brooke MH. Muscle biopsy: a modern approach. Philadelphia: WB Saunders, 1973:1-475. 19. Cormier V, Rustin P, Bonnefond JP, et al. Hepatic failure in disorders of oxidative phosphorylation with neonatal onset. J PEDIATR 1991;119:951-4. 20. Bourgeron T, Chretien D, R6tig A, Munnich A, Rustin P. Prenatal diagnosis of cytochrome c oxidase deficiency in cultured amniocytes is hazardous. Prenat diagn 1992;12:548-9. 21. G6rard B, Bourgeron T, Chretien D, R6tig A, Munnich A, Rustin P. Uridine preserves the expression of respiratory enzyme deficiencies in cultured fibroblasts. Eur J Pediatr 1992;152:270. 22. Sidi D, LeBidois J, Pi6chaud JF, et al. Activit6s enzymatiques de la cha~ne respiratoire mitochondriale dans les myocardiopathies de l'enfant: &ude prospective de 34 cas par biopsies endomyocardiques. Arch Mal Coeur Vaiss 1992;85:541-6. 23. Capaldi RA, Halphen DG, Zhang YZ, Yanamura W. Complexity and tissue specificity of the mitochondrial respiratory chain. J Bioenerg Biomembr 1988;20:291-311.