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Cardiomyopathy and angiopathy in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes
Cardiomyopathy and angiopathy in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes In four patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) in which mutated mitochondrial deoxyribonucleic acid was sea% hypertrophic cardiomyopathy and angiopathy was demonstrated by echocardiography, dipyridamole stress scintigraphy, and cardiac catheterization. On stress scintigraphy with dipyridamole, three patients showed hypoperfusion in the early image and a “filling-in” pattern in the late image. However, coronary angiography did not demonstrate narrowing of the large vessels in these patients. Light and electron microscopy of endomyocardial biopsy specimens indicated abnormal mitochondrla, with marked increase in the number and size of mitochondria in endothelium. Modlied Gomori’s trichrome staining in biopsied endomyocardiil specimens revealed a red-purple deposit similar in appearance of the ragged-red fibers in skeletal muscle, a characteristic finding of mitochondrial disease. Deterioration of complex I in the mitochondrial electron transfer system, which is widely observed in various mitochondrial diseases, appeared in biipsfed skeletal muscle of our patients, indicating deficiency of some subunits of complex I. These results indite that mitochondrial diseases such as MELAS show not only cardiomyopathy but also angiopathy. We speculate that proliferation of mitochondria leads to narrowing of the lumen of arterioles, which might be responsible for the ischemic findings observed scintigraphically. (AM HEART J 1994;128:733-41.)
Wataru Sate, MD, PhDF Masashi Tanaka, MD, PhD,b Satoru Sugiyama, Taisuke Nemoto, MD,a Kenji Harada, MD,a Yasunori Miura, MD,a Yasuko Kobayashi, MD,a Atsuko Goto, MD,a Goro Takada, MD, PhD,” and Takayuki Ozawa, MD, PhDb AKita and Nugoya, Japan
Because cellular energy supply depends on the proper functioning of the mitochondrial energy-producing system, failing mitochondria may lead to various diseases. The molecular biologic definition of primary mitochondrial disease is a genetic defect in mitochondrial enzymes or translocators. Mitochondria have their own deoxyribonucleic acid (mtDNA), which encodes 13 subunits of the five-complex enerFrom the aDepartment and the bDepartment versity of Nagoya.
of Pediatrics, of Biomedical
Akita University School of Medicine; Chemistry, Faculty of Medicine, Uni-
Supported in part, by a Grants-in-Aid for General Science Research (62570128) to (Dr. Tanaka) and for Scientific Research on Priority Areas (Bioenergetics, 01617002) to (Dr. Ozawa) from the Ministry of Education, Science, and Culture of Japan; and by Grant 01-02-39 to (Dr. Ozawa) from the Ministry of Health and Welfare Japan. Received
for publication
July
22, 1993;
accepted
Jan.
12,1994.
Reprint requests: Takayuki Ozawa, MD, PhD, Department of Biomedical Chemistry, Faculty of Medicine, University of Nagoya, Tsuruma, Showaku, Nagoya 466, Japan. Copyright P 1994 0002-8703/94/$3.00
by Mosby-Year + 0 4/l/57044
Book,
Inc.
MD, PhD,h
gy-producing system. Other subunits are encoded by nuclear DNA. MtDNA is known to be highly vulnerable compared with nuclear DNA,l and hence is of principal importance in the genesis of primary mitochondrial disease. Mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) comprise one of the primary mitochondrial diseases and constitute a clinical syndrome characterized by muscle weakness, recurrent episodic vomiting, headache, and strokelike episodes such as hemianopia and cortical blindness.2 Molecular biologic analysis has revealed a mutation in the mitochondrial tRNALeuCUUR) gene coexisting with wild type MtDNA3-5 in these patients. This mutation is believed to lead to dysfunction of the mitochondrial electron-transducing system.6 Accumulation of morphologically abnormal mitochondria, which were found in various tissues, including cardiac muscles, has been described.7 Although cardiac abnormalities such as hypertrophic cardiomyopathy are often encountered in patients with MELAS, coronary angi733
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lebfe I. ClinicaJ and iaboratory features Patient
Age on admission (yr) Sex Weakness Seizure Dementia Short stature Episodicvomiting Corticalblindness Hemiparesis or hemisnopia Sensorineural hearingloss Lactic acidosis Family history Ragged-red fibers Low densityareain brain CT scan +, Present;
1
2
3
4
19
13
13
10
F t
M +
M -
F +
-I-
+
-I-
-
4 +
+ +
-
+
+ + + + +
t t
+ + + + +
+ + + t +
+ -
-, absent.
opathy has rarely been observed. We report on cardiomyopathy and/or cardioangiopathy detected in four MELAS patients with echocardiography, dipyridamole stress scintigraphy, and cardiac catheterization. Biochemical and/or immunologic analysis of skeletal muscle from the patients is also described.
Our results indicate that mutations in cardiomyopathy and angiopathy MELAS.
of mtDNA result in patients with
METHODS Patients. The clinical and laboratory features of four patients with MELAS are summarizedin Table I. The ages of the patients at the time of the cardiac studies are given. Skeletal muscle specimenswere obtained from four patients by open biopsy after permissionand informed consent were given. Patient 1 died at the ageof 22 years, and other patients are alive. There wasno history of hypertension, diabetes mellitus, or hypercholesterolemia in these four patients. Cardiac examination. All patients underwent right heart catheterization with a triple-lumen thermistor SwanGanz catheter to measurepulmonary artery pressure,pulmonary artery wedgepressure,and cardiac output. A thermodilution technique was used to determine cardiac output. Selective coronary arteriography and left ventriculography were alsoperformed, and each coronary artery was viewed in multiple projections in all patients. Left ventricular volume in each patient was calculated by biplane cineangiography,and left ventricular ejection fraction wasdetermined by the area length method. Coronary and left ventricular angiographic data were analyzed according to the method of the American Heart Association Committee Report.8 M-mode echocardiogramsof the left ventricle were obtained with an ultrasound system (Toshi-
October 1994 Heart Journal
ba Carp, Tokyo, Japan) and 3.5 MHz transducer. We measured interventricular septal thickness (IVS) and left ventricular posterior wall thickness (LVPW). In each patient a pulsedDoppler examination of the left ventricular inflow tract was performed immediately after M-mode echocardiography. The Doppler samplevolume waspositioned in the mitral valve annuluswith the apicallong-axis view. The position of the sample volume was adjusted so that the Doppler beamwas aligned parallel to the flow as much as possible.Electrocardiograms and the respiratory curve of eachpatient were recordedsimultaneously with a pressure transducer placed on the abdomen.All examinations were recorded at a paper speedof 50 mm/set. With the aid of a computer-interfaced digital pad, the following parameters were measured:peak velocity of early passivefilling period (E) and late atrial filling period (A), integrated velocities for total E and A filling, and heart rate (HR). From these measurementswe calculated the peak E/A ratio and the ratio of E and A integrated velocities, Patients exercisedon a treadmill, and 3 mCi of thallium-201 was administered 5 min after the completion of dipyridamole infusion. Single photon emissioncomputed tomographic (SPECT) images were obtained with a wide-field-of-view rotating gamma cameraequipped with a long-energy, high-resolution, parallel-hole collimator (Toshiba GCA/GOl E, Tokyo, Japan). The SPECT imageswere acquired at the 80keV photopeak of thallium-201 with a symmetric 10% window. The early imagesand delayed imaging obtained 10 to 15 min or 4 hours after thallium-201 administration, respectively, were analyzed by two observerswho had no knowledge of the patients’ clinical history and coronary angiographic data. Restriction enzyme analysis of mtDNA. Total DNA was extracted from 10 ml of whole blood by the phenol/chloroform method asreported previously.gAn mtDNA fragment of 800basepair (nucleotide position 2881to 3680)wasamplified with polymerasechain reaction primers, L288 (5’CTACTATACT CAATTGATCC-3’) and H366 (5’GAGTTTGATG CTCACCCTGA-3’), after 30 cycles of the following conditions: 15 set of denaturation at 94” C, 15set of annealingat 55” C, and 40 set of primer extension at 72OC. The polymerasechain reaction product wasprecipitated with ethanol and wasdigested by the restriction enzyme Apa I. Biochemical studies. Mitochondria were isolated from biopsied skeletal muscles according to the method of Bookelman et al. loEnzymic activities of rotenone-sensitive nicotinamide adenine dinucleotide (NADH)-ubiquinone oxidoreductase (complex I), succinate-ubiquinone oxidoreductase (complex II), succinate-cytochrome c reductase (complex II-III), ubiquinol-cytochrome c oxidoreductase(complex III), cytochrome c oxidase(complex IV), and oligomycin-sensitive adenosine triphosphatase (complex V) were measuredas reported previously.” In patients 1 and 2, subunits of complexesI, II, III, IV, and V were analyzed by the Western blot method with specific antibodiesagainstbovine holoenzymesaccordingto the method of Tanaka et all2 Antibody binding was detected with iodine 125labeled protein A (Amersham,U. K.). Protein was
Vduma 120, Number 4 American Heart Journal
Sat0 et al.
Table II. Summary of cardiac examination
__ .-.Patient
Cardiothoracic EGG
No murmur
Systolic murmur
Auscultation ratio (% )
Echocardiogram IVS (mm) LVPW (mm) Scintigraphy Hypoperfusion in early image “Filling in” in redistribution image mPA (mm Hg) LV (mm Hg) A0 (mm Hg) LVEDVI (ml/m2) CI (Llminlm? EF Coronary angiography
66 B-type WPW syndrome
47 Flattened T waves in V5 and V6
12 12
34/16 130/4,
735
__.--
Midsystolic murmur 45 Normal
No nntrmur 47 Flattened and negative T waves in II, III, aVF. V.i. and V6 I(i (8
7.5 7.5
10 10
-i-
-
+
t
+
-
+
+
1‘221
EDP 18 136/85 11111 56 4.0
0.86 Normal
22/10
[15]
9114, EDP 14 92/61 [76] 96 5.3 0.70
Normal
[ll] 95/Q, EDP 10
22/7
42/25
96/59
112/O. EDP 9 118/81 [ 1011
1791 88 5.4 0.77
Normal
[33]
5.; 3.7 Q.56
Normal
;J,M ean value; IVS, intraventricnlar septum (normal range 5.8 i 0.7 mm); LVPW, left ventricular posterior wall (normal range 5.9 -c 0.8 nun); scintigraphy, stressacintigraphy with dypiridamole; f, present; -, absent; mPA, main pulmonary artery; LV, left.ventricle; EDP, end-diastolic pressure; AO, aorta; LVEDVI, left ventricular end diastolic volume index (normal range 73 + 11 ml/m’; Cl, cardiac index; EF, ejection fraction.
measured by the bicinchoninic acid method.13 For the control, autopsied tissue of age-matched patients wasobtained within 2 hours after death. The activity of each of five complexes was also determined in these tissues. Morphologic studies. Right ventricular endomyocardial specimens were frozen in liquid-cooled isopentane and cross-sectioned to a thickness of 8 pm in a cryostat for histochemical staining. The specimens were then stained by the elastica Masson method and Gomori’s trichrome staining. The electron microscopic studies of endomyocardial specimens were processed as described by Kobayashi et a1.14This study was performed according to the declaration of Helsinki. RESULTS
All patients were clinically diagnosed as having MELAS, and an A-to-G transition mutation at nucleotide 3,243 (Cambridge number15) was detected in mtDNA in white blood cells or autopsied tissues by restriction enzyme analysis of mtDNA. In patient 1, mtDNA was detected in all of the autopsied organs, including cardiac muscle. Cardiac studies with echocardiography and stress myocardial scintigraphy with dipyridamole are shown in Table II. Cardiac catheterization studies indicated normal systolic function and normal coronary angiography. Moderate hypertrophy of the cardiac
muscles was found by M-mode echocardiography; decreases in E, integrated velocity for total E, and E/A ratio obtained from a pulsed Doppler examination showed decreased diastolic left ventricular function. In dipyridamole stress scintigraphy arterial insufficiency, that is, hypoperfusion in the early image and a “filling in” pattern in the late image, was found in the small vessels of patients 1,3, and 4. These cardiac studies indicated the earliest signs of cardiac failure resulting from hypertrophic cardiomyopathy and cardioangiopathy. Findings of SPECT images and those of coronary angiography of a representative case (patient 1) are shown in Figs. 1 and 2, respectively. Activities of the enzyme complexes in mitochondria from the skeletal muscles of four patients are shown in Table III. The enzymatic activities of complex I were markedly decreased in all patients (2 % to 27% of the mean control value). The enzymatic activities of complexes II and V in patients 1 and 2 were about twice as high as the normal values. The enzymatic activities of the other complexes were within the normal range. Immunoblot analysis of the subunits in patients 1 and 2 are shown in Fig. 3. The amounts of complex I subunits, particularly of subunit 75 kDa, were decreased in the mitochondria
736
Sato et al.
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October 1994 Heart Journal
Fig. 1. Coronary angiography of patient 1. No stenosiswas observed in coronary artery.
Fig. 2. SPECT imagesin dipyridamole stressscintigraphy of patient 1. Hypoperfusion (arrow) in early image {A and 6) and a “filling-in” pattern in the late image (C and D) were found.
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Number 4 Heart Journal
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Fig. 3. Western blot analysis of subunits of complexes I, II, III, IV, and V. C, Control mitochondria (15 pg protein); P1, patient 1 mitochondria (15 rg protein); P2, patient 2 mitochondria (15 g protein); I, complex 1 (figures show molecular masses (KDa) of detected subunits of complex I); ZZ,complex II (figure shows molecular mass of subunit); III, complex III (Core, cl, and ISP show core proteins, cytochrome cl and ironsulfur protein); ZV, complex IV (2, 4, 5, 6, and 7 show subunits 2, 4, 5, 6, and 7); V, complex V (LYand 0 show subunits (Yand (3).
Ill. Enzymatic activities in mitochondria from skeletal muscle
Table
---
-..__-
Patient Complex I II II-III III IV V Activities
are expressed
1
2
3
4
3 (2) 672 (212)
36 (25) 765 (241)
36 (25)
39 (27)
102 (70)
157 (108)
607 (83) 457 (75) 148 (202)
397 (54) 421 (67) 156 (214)
958 (156) 59 (81)
730 (119) 83 (114)
in nmol/min/mg
of mitochondrial
protein.
Figures in parentheses
from the patients. The amounts of complex II subunits were increased, which is consistent with the results of enzymatic activity measurement, The amounts of complexes III, IV, and V were normal or slightly decreased. Light microscopy of the endomyocardial specimen from patient 1 revealed disorganization of the myofibrillar architecture, interstitial fibrosis, and vacuolization of myocardial cells in elastica Masson staining. Modified Gomori’s trichrome staining of the endomyocardial specimen from patient 4 showed a red-purple deposit mainly around the nucleus similar in appearance to the ragged-red fibers in the skeletal muscle (Fig. 4). Electron microscopy revealed increased numbers and size of mitochondria, concen-
are percentages
Control (mean 2 SD) 146 317 145 731 613 73
+ tk t k ?I
59 64 59 319 276 22
of mean control
Range 82-246 216-350 42-243 411-1117 325-l 143 30-97
values
tric configuration of cristae, decreased myofibrils (Fig. 5), and swollen endothelial cells, resulting in narrowing of the capillary lumen. These findings in heart muscle by light and electron microscopy were similar to those found in skeletal muscle. DISCUSSION
Recent advances in molecular medicine have emphasized that mutations in mtDNA are of primary importance in the development of mitochondrial dysfunction, although the mitochondrial energytransducing system is also vulnerable to various environmental factors such as malnutrition, ischemia, hormonal disturbances,16 drugs,l’ and poisoning.lsy I9 It has previously been reported that hypertrophic
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et al.
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Fig. 4. Modified Gomori’s trichrome staining of biopsied endomyocardial specimen from patient 4. Redpurple deposit around nucleus is similar to appearance of ragged-red fibers in skeletal muscle.
cardiomyopathy is associated with dysfunction of the mitochondrial electron-transfer system,2o-22 and deletion of mtDNA and several point mutations in mtDNA have been reported in patients with hypertrophic cardiomyopathy.23$ 24 These findings suggest that genetic disturbance of the mitochondrial energy-transducing system is an important factor in hypertrophy of cardiac muscle. In the present study abnormal mitochondria in endomyocardial specimens were observed morphologically. Mitochondria carry out the Krebs cycle and ,&oxidation pathway for fatty acids. These degenerative sequences essentially remove hydrogen from metabolic fuels with the release of carbon dioxide and transfer it via coenzymic carriers to the mitochondrial respiratory chain. The chain then transfers these reducing equivalents, eventually to react with molecular oxygen in the production of water. Reducing equivalents, NADH or succinate, are transferred via complex I, NADHubiquinone oxidoreductase, or via complex IT, succinate-ubiquinone oxidoreductase, respectively; to complex III, ubiquinol-cytochrome c oxidoreductase; and then to complex IV, cytochrome c oxidase, as shown in Fig. 6. These complexes, together with
complex V, adenosine triphosphatase, are responsible for the overall process of oxidative phosphorylation, i.e., energy production. In the present cases the activity of complex I was decreased and the activity of complex II was increased. Deterioration of complex I activity might induce this compensatory change. Because deterioration of complex I was observed in mitochondrial neuromyopathy6 and Parkinson’s disease,25 and because various agentslgp 26 and agingz7 also show detrimental effect to the activity of complex I, the role of complex I might be emphasized in the genesis of mitochondrial disease. Complex I is the largest of the respiratory chain complexes.and seems to be the most vulnerable to disturbance of membrane structural integrity. In addition, the vulnerability of mtDNA is expected to be much higher than the vulnerability of nuclear DNA.’ Because mtDNA encodes 7,3,2,1 and no subunits of complexes I, IV, V, IIT, and II, respectively, the effect of mutation of mtDNA on the vulnerability of complex I may be larger than that on other complexes. In the present study, biochemical analysis demonstrated markedly decreased activities of complex I in biopsied skeletal muscles. Immunochemical analysis
volume 128, Number 4 American Hod Journal
Sat0 et al.
739
Fig. 5. Electron micrograph showsA, Giant mitochondria (right); B, vacuolization of mitochondria fleftl in biopsied endomyocardial specimenfrom patient 1.
Complex I NADH
I.3 Cyt. a
02
% a3
Complex II
Complex Ii I
Complex IV
6. Schematicrepresentation of mitochondrial electron transfer system.FE, acid extractable Aavin; Fs, acid nonextractable flavin; nH Fe, nonhemeiron; Cyt, cytochrome; Cu, copper.
Fig.
also indicated
a decreased amount
of complex I sub-
units in cardiac muscles. An A-to-G transition at 3,243 in the tRNALeU(UUR) gene was observed in all four cases studied. This mu-
tation has been reported to cause an impairment of mtDNA-encoded protein synthesis,2” and this mutation results in severe impairment of 16s rRNA tranbiologic scription termination. 2g The molecular mechanism responsible for proliferation of mito-
chondria with mutated mtDNA remains obscure, however, and further investigations are required for clarification.
In addition to hypertrophic
cardiomy-
opathy, mtDNA mutations at 3,243 are often associated with the Wolff-Parkinson-White syndrome.24T 3o We also found mtDNA mutations in patients with conduction block.31 These results suggest that mtDNA mutations may be implicated as a cause of conduction disturbances.
740
Sat0 et al.
Involvement of mtDNA mutation in angiopathy has rarely been reported. In evaluation of patients with stress scintigraphy with dipyridamole, three patients showed ischemia-like changes. Coronary angiography, however, did not demonstrate narrowing of the large vessels in these three patients. Therefore this arterial insufficiency might be the result of lesions in smal1 arterial vessels of the heart. Indeed, we observed marked increase in the number and size of mitochondria in endothelium and narrowed lumen of the arterioles. Ohama et a1.7observed similar findings in cerebral arteries of patients with MELAS, where a striking increase in the number of mitochondria in smooth muscle and endothelial cells occurred. On the basis of our results, we speculate that some patients with ischemic ST-T segment changes seen electrocardiographically but without narrowing of the large vessels angiographically might have angiopathy from mtDNA mutations. There are two or three copies of mtDNA in each mitochondrion, and several thousandmtDNA may exist in a ce11.32It is known that mtDNA with mutation is not distributed uniformly in the tissue33 and that tissue having mtDNA with mutation over a certain level might be expected to show deleterious function. These results present a challenge to the traditional treatment in those patients who have previously been considered to have arteriosclerosis. In addition, as seen in patient 3, hypoperfusion found on stress scintigraphy in a patient without cardiac hypertrophy can occur, indicating that angiopathy can be seen independently of cardiac hypertrophy. In conclusion, cardiomyopathy and/or angiopathy may be observed in patients with mtDNA mutation, and it is possible that other formal coronary artery disease such as arteriosclerosis may have an underlying mtDNA mutation. This possibility warrants further mtDNA study in coronary artery disease. We thank M. Bodman. our department language consultant, for reading the previous draft and making suggestions on language and style. REFERENCES
Linnane AW, Marzuki S, Ozawa T, Tanaka M. Mitochondrial DNA mutations as an important contributor to aging and degenerative diseases. Lancet 1989;1:642-5. Pavlakis SG, Phillips PC, DiMauro S, De Vito DC, Rowland LP. Mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes: a distinctive clinical syndrome. Ann Neurol 1984;16:481-8. Goto Y, Nonaka I, Horai S. A mutation in the tRNALeu(U”R) gene associated with the MELAS subgroup of mitochondrial encepholomyopathies. Nature 1990;348:651-3. Kobayashi Y, Momoi MY, Tominaga K, Momoi T, Nihei K, Yanagisawa M, Kagawa Y, Ohta S. A point mutation in the mitochondrial tRNALeu(UUR) gene in MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). Biochem Biophys Res Commun 1996;173:816-22.
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5. Tanaka M, Ino H, Ohno K, Ohbayashi T, Ikebe S, Sano T, Ichiki T, Kobayashi M, Wada Y, Ozawa T. Mitochondrial DNA mutations in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Biochem Biophys Res Commun 1991;174:861-8. 6. Ichiki T, Tanaka M, Nishikimi M, Suzuki H, Ozawa T, Kobayashi M, Wada Y. Deficiency of subunits of complex 1 and mitochondrial encephalomyopathy. Ann Neurol 1988;23:28794. 7. Ohama E, Ohara S, Ikuta F, Tanaka K, .Nishizawa M, Miyatake T. Mitochondrial angiopathy in cerebral blood vessels of mitochondrial encephalomyopathy. Acta Neuropathol 1987;74:226-33. 8. A.H.A. Committee Report. A reporting system on patients evaluated for coronary artery disease. Circulation 1975;51:540. 9. Ota Y, Tanaka M, Sato W, Ohno K, Yamamoto T, Maehara M, Negoro T, Watanabe K, Awaya S, Ozawa T. Detection of platelet mitochondrial DNA deletions in Kearns-Sayre syndrome. Invest Ophthalmol Vis Sci 1991;32:2667-75 10. Bookelman H, Trijbels JMF, Sengers RCA, Janssen AJM. Measurement of cytochromee in human skeletal muscle mitochondria, isolated from fresh and frozen stored muscle specimens. Biochem Med 1978;19:366-73. 11. Yoneda M, Tanaka M, Nishikimi M, Suzuki H, Tanaka M, Nishizawa M, Atsumi T, Ohama E, Horai S, Ikuta F, Miyatake T, Ozawa T. Pleiotropic molecular defects in energy-transducing complexes in mitochondrial encephalomyopathy (MELAS). J Neurol Sci 1989;92:143-58. 12. Tanaka M, Miyabayashi S, Nishikimi M, Suzuki H, Shimomura Y, Ito K, Narisawa K, Tada K, Ozawa T. Extensive defects of mitochondrial electron-transfer chain in muscular cytochrome c oxidase deficiency. Pediatr Res 1988;24:447-54. 13. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH. Provenzano MD. Fuiimoto EK. Goeke NM. Olson BJ. Klenk DC. Measurement of protein using bicinchbninic acid: Anal Biochem 1985;150:76-85. 14. Kobayashi Y, Miyabayashi S, Takada G, Narisawa K, Tada K, Yamamoto TY. Ultrastructural study of the childhood mitochondrial myopathic syndrome associated with lactic acidosis. Eur J Pediatr 1982;139:25-30. 15. Anderson S, Bankier AT, Barrel1 BG, de Bruijn MHL, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJH, Staden R, Young IG. Sequence and organization of human mitochondrial genome. Nature 1981; 290:457-65. 16. Sugiyama S, Kato T, Ozawa T, Yagi K. Deterioration of mitochondrial function in heart muscles of rats with hypothyroidism. J Clin Biochem Nutr 1991:11:199-204. 17. Ogawa Y, Kondo T, Sugiyama S, Ogawa K, Satake T, Ozawa T. Role of phosphotipase in the genesis of doxorubicininduced cardiomyopathy in rats. Cancer Res 1987;47: 1239-43. 18. Kato T, Sugiyama S, Hanaki Y, Fukushima A, Akiyama N, Ito T, Ozawa T. Role of acetylchohne in pyridostigmine-induced myocardial injury: possible involvement of parasympathetic nervous system in the genesis of cardiomyopathy. Arch TOXicol 1989;63:137-43. 19. Sugiyama S, Takasawa M, Hayakawa M, Esumi H, Ozawa T. Detrimental effects of 2-amino-1-methyl-6-phenylimidaxo[4,5-blpyridine, a mutagenic agent, on mitochondrial respiration among various rat tissues. Biochem Mol Biol Int 1993; 30:797-805. 20. Papadimitriou A, Neustein HB, DiMauro S, Stanton R, Bresolin N. Histiocytoid cardiomyopathy of infancy: deficiency of reducible cytochrome b in heart mitochondria. Pediatr Res 1984;18:1023-8. 21. Zeviani M, VanDyke DH, Servidei S, Bauserman SC, Bonilla E, Beaumont ET, Sharda J, VanderLaan K, DiMauro S. Myopathy and fatal cardiopathy due to cytochrome c oxidase deficiency. Arch Neurol 1986;43:1198-202. 22. Smeitink JAM, Sengers RCA, Trijbels JMF, Ruitenbeek W,
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Da&Is 0, Stadbouders AM, Kock-Jansen MJH. Fatal cardiomyopathy associated with cataract and mitochondrial myopathy. Eur J Pediatr 1989;148:656-9. 23. Hattori K, Ogawa T, Kondo T, Mochizuki M, Tanaka M, Sugiyama S, Ito T, Satake T, Ozawa T. Cardiomyopathy with mitochondrial DNA mutations. AM HEART J 1991;122:866-9. 24. Obayashi T, Hattori K, Sugiyama S, Tanaka M, Itoyama S, Deguchi H, Kawamura K, Koga Y, Toshima H, Takeda N, Nagano M, Ito T, and Ozawa T. Point mutations in mitochondrial DNA in patients with hypertrophic cardiomyopathy. AM HEART
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25. Hattori N, Tanaka M, Ozawa T, Mizuno Y. ImmunohistochemicaI studies on complex I, II, III, and IV of mitochondria in Parkinson’s disease. Ann Neural 1991;30:563-71. 26. Mizuno Y, Suzuki K, Sone N, Saitoh T. Inhibition of mitocbondrial respiration by l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in muuse brain in vivo. Neurosci Lett 1988;91:349-53. 27. Takasawa M, Hayakawa M, Sugiyama S, Ozawa T. Age-associated damage in mitochondrial function in rat heart. Esp GerontoI 1993;28:269-80. 28. King MP, Koga Y, Davidson M, Schon EA. Defects in mitochondrial protein synthesis and respiratory chain activity
.%I!:,
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segregate with the tRNALeu(liUR) mutation associated with mitochandrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes. Mol Cell Biol 1992;12:480-90. 29. Hess JF, Parisi MA, Bennett JL, Clayton DA. Impairment of mitochondrial transcription termination by a point mutation associated with the MELAS subgroup of mitochondrial encephalomyopathies. Nature 1991;351:236-9. 30. Zeviani M, Gellera C, Antozzi C, Rimoldi M, Morandi I,, VilIani F. Tiranti V. DiDonato S. Maternallv inherited mvonathv and cardiomyopathy: association with mutation in mitochondrial DNA tRNAL”U’U”R’. Lancet 1991;338:143-7. 31. Sato W, Tanaka M, Sugiyama S, Hattori K, Ito T, Kawaguchi H, Onozuka H, Yasuda H, Ito K, Takada G, Ozawa T. Deletion of mitochondriai DNA in a patient with conduction block. I
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32. Robin ED, W ong R. Mitochondrial DNA molecules and virtual number of mitochondria per ceII in mammalian cells. J Cell Physiol 1988;136:50’7-13. 33. Ozawa T, Tanaka M, Sato W, Ohno K, Sugiyama S, Yoneda M, Yamamoto T, Hattori K, Ikebe S, Tashiro M, Sahashi K. Mitochondrial DNA mutations as an etiology of human degenerative diseases. In: Ozawa T, Kim CH. eds. Bioenergetits. New York: Plenum Press, 1990:413-27.
Wataru Sate, MD, PhDF Masashi Tanaka, MD, PhD,b Satoru Sugiyama, Taisuke Nemoto, MD,a Kenji Harada, MD,a Yasunori Miura, MD,a Yasuko Kobayashi, MD,a Atsuko Goto, MD,a Goro Takada, MD, PhD,” and Takayuki Ozawa, MD, PhDb AKita and Nugoya, Japan
Because cellular energy supply depends on the proper functioning of the mitochondrial energy-producing system, failing mitochondria may lead to various diseases. The molecular biologic definition of primary mitochondrial disease is a genetic defect in mitochondrial enzymes or translocators. Mitochondria have their own deoxyribonucleic acid (mtDNA), which encodes 13 subunits of the five-complex enerFrom the aDepartment and the bDepartment versity of Nagoya.
of Pediatrics, of Biomedical
Akita University School of Medicine; Chemistry, Faculty of Medicine, Uni-
Supported in part, by a Grants-in-Aid for General Science Research (62570128) to (Dr. Tanaka) and for Scientific Research on Priority Areas (Bioenergetics, 01617002) to (Dr. Ozawa) from the Ministry of Education, Science, and Culture of Japan; and by Grant 01-02-39 to (Dr. Ozawa) from the Ministry of Health and Welfare Japan. Received
for publication
July
22, 1993;
accepted
Jan.
12,1994.
Reprint requests: Takayuki Ozawa, MD, PhD, Department of Biomedical Chemistry, Faculty of Medicine, University of Nagoya, Tsuruma, Showaku, Nagoya 466, Japan. Copyright P 1994 0002-8703/94/$3.00
by Mosby-Year + 0 4/l/57044
Book,
Inc.
MD, PhD,h
gy-producing system. Other subunits are encoded by nuclear DNA. MtDNA is known to be highly vulnerable compared with nuclear DNA,l and hence is of principal importance in the genesis of primary mitochondrial disease. Mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) comprise one of the primary mitochondrial diseases and constitute a clinical syndrome characterized by muscle weakness, recurrent episodic vomiting, headache, and strokelike episodes such as hemianopia and cortical blindness.2 Molecular biologic analysis has revealed a mutation in the mitochondrial tRNALeuCUUR) gene coexisting with wild type MtDNA3-5 in these patients. This mutation is believed to lead to dysfunction of the mitochondrial electron-transducing system.6 Accumulation of morphologically abnormal mitochondria, which were found in various tissues, including cardiac muscles, has been described.7 Although cardiac abnormalities such as hypertrophic cardiomyopathy are often encountered in patients with MELAS, coronary angi733
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lebfe I. ClinicaJ and iaboratory features Patient
Age on admission (yr) Sex Weakness Seizure Dementia Short stature Episodicvomiting Corticalblindness Hemiparesis or hemisnopia Sensorineural hearingloss Lactic acidosis Family history Ragged-red fibers Low densityareain brain CT scan +, Present;
1
2
3
4
19
13
13
10
F t
M +
M -
F +
-I-
+
-I-
-
4 +
+ +
-
+
+ + + + +
t t
+ + + + +
+ + + t +
+ -
-, absent.
opathy has rarely been observed. We report on cardiomyopathy and/or cardioangiopathy detected in four MELAS patients with echocardiography, dipyridamole stress scintigraphy, and cardiac catheterization. Biochemical and/or immunologic analysis of skeletal muscle from the patients is also described.
Our results indicate that mutations in cardiomyopathy and angiopathy MELAS.
of mtDNA result in patients with
METHODS Patients. The clinical and laboratory features of four patients with MELAS are summarizedin Table I. The ages of the patients at the time of the cardiac studies are given. Skeletal muscle specimenswere obtained from four patients by open biopsy after permissionand informed consent were given. Patient 1 died at the ageof 22 years, and other patients are alive. There wasno history of hypertension, diabetes mellitus, or hypercholesterolemia in these four patients. Cardiac examination. All patients underwent right heart catheterization with a triple-lumen thermistor SwanGanz catheter to measurepulmonary artery pressure,pulmonary artery wedgepressure,and cardiac output. A thermodilution technique was used to determine cardiac output. Selective coronary arteriography and left ventriculography were alsoperformed, and each coronary artery was viewed in multiple projections in all patients. Left ventricular volume in each patient was calculated by biplane cineangiography,and left ventricular ejection fraction wasdetermined by the area length method. Coronary and left ventricular angiographic data were analyzed according to the method of the American Heart Association Committee Report.8 M-mode echocardiogramsof the left ventricle were obtained with an ultrasound system (Toshi-
October 1994 Heart Journal
ba Carp, Tokyo, Japan) and 3.5 MHz transducer. We measured interventricular septal thickness (IVS) and left ventricular posterior wall thickness (LVPW). In each patient a pulsedDoppler examination of the left ventricular inflow tract was performed immediately after M-mode echocardiography. The Doppler samplevolume waspositioned in the mitral valve annuluswith the apicallong-axis view. The position of the sample volume was adjusted so that the Doppler beamwas aligned parallel to the flow as much as possible.Electrocardiograms and the respiratory curve of eachpatient were recordedsimultaneously with a pressure transducer placed on the abdomen.All examinations were recorded at a paper speedof 50 mm/set. With the aid of a computer-interfaced digital pad, the following parameters were measured:peak velocity of early passivefilling period (E) and late atrial filling period (A), integrated velocities for total E and A filling, and heart rate (HR). From these measurementswe calculated the peak E/A ratio and the ratio of E and A integrated velocities, Patients exercisedon a treadmill, and 3 mCi of thallium-201 was administered 5 min after the completion of dipyridamole infusion. Single photon emissioncomputed tomographic (SPECT) images were obtained with a wide-field-of-view rotating gamma cameraequipped with a long-energy, high-resolution, parallel-hole collimator (Toshiba GCA/GOl E, Tokyo, Japan). The SPECT imageswere acquired at the 80keV photopeak of thallium-201 with a symmetric 10% window. The early imagesand delayed imaging obtained 10 to 15 min or 4 hours after thallium-201 administration, respectively, were analyzed by two observerswho had no knowledge of the patients’ clinical history and coronary angiographic data. Restriction enzyme analysis of mtDNA. Total DNA was extracted from 10 ml of whole blood by the phenol/chloroform method asreported previously.gAn mtDNA fragment of 800basepair (nucleotide position 2881to 3680)wasamplified with polymerasechain reaction primers, L288 (5’CTACTATACT CAATTGATCC-3’) and H366 (5’GAGTTTGATG CTCACCCTGA-3’), after 30 cycles of the following conditions: 15 set of denaturation at 94” C, 15set of annealingat 55” C, and 40 set of primer extension at 72OC. The polymerasechain reaction product wasprecipitated with ethanol and wasdigested by the restriction enzyme Apa I. Biochemical studies. Mitochondria were isolated from biopsied skeletal muscles according to the method of Bookelman et al. loEnzymic activities of rotenone-sensitive nicotinamide adenine dinucleotide (NADH)-ubiquinone oxidoreductase (complex I), succinate-ubiquinone oxidoreductase (complex II), succinate-cytochrome c reductase (complex II-III), ubiquinol-cytochrome c oxidoreductase(complex III), cytochrome c oxidase(complex IV), and oligomycin-sensitive adenosine triphosphatase (complex V) were measuredas reported previously.” In patients 1 and 2, subunits of complexesI, II, III, IV, and V were analyzed by the Western blot method with specific antibodiesagainstbovine holoenzymesaccordingto the method of Tanaka et all2 Antibody binding was detected with iodine 125labeled protein A (Amersham,U. K.). Protein was
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Sat0 et al.
Table II. Summary of cardiac examination
__ .-.Patient
Cardiothoracic EGG
No murmur
Systolic murmur
Auscultation ratio (% )
Echocardiogram IVS (mm) LVPW (mm) Scintigraphy Hypoperfusion in early image “Filling in” in redistribution image mPA (mm Hg) LV (mm Hg) A0 (mm Hg) LVEDVI (ml/m2) CI (Llminlm? EF Coronary angiography
66 B-type WPW syndrome
47 Flattened T waves in V5 and V6
12 12
34/16 130/4,
735
__.--
Midsystolic murmur 45 Normal
No nntrmur 47 Flattened and negative T waves in II, III, aVF. V.i. and V6 I(i (8
7.5 7.5
10 10
-i-
-
+
t
+
-
+
+
1‘221
EDP 18 136/85 11111 56 4.0
0.86 Normal
22/10
[15]
9114, EDP 14 92/61 [76] 96 5.3 0.70
Normal
[ll] 95/Q, EDP 10
22/7
42/25
96/59
112/O. EDP 9 118/81 [ 1011
1791 88 5.4 0.77
Normal
[33]
5.; 3.7 Q.56
Normal
;J,M ean value; IVS, intraventricnlar septum (normal range 5.8 i 0.7 mm); LVPW, left ventricular posterior wall (normal range 5.9 -c 0.8 nun); scintigraphy, stressacintigraphy with dypiridamole; f, present; -, absent; mPA, main pulmonary artery; LV, left.ventricle; EDP, end-diastolic pressure; AO, aorta; LVEDVI, left ventricular end diastolic volume index (normal range 73 + 11 ml/m’; Cl, cardiac index; EF, ejection fraction.
measured by the bicinchoninic acid method.13 For the control, autopsied tissue of age-matched patients wasobtained within 2 hours after death. The activity of each of five complexes was also determined in these tissues. Morphologic studies. Right ventricular endomyocardial specimens were frozen in liquid-cooled isopentane and cross-sectioned to a thickness of 8 pm in a cryostat for histochemical staining. The specimens were then stained by the elastica Masson method and Gomori’s trichrome staining. The electron microscopic studies of endomyocardial specimens were processed as described by Kobayashi et a1.14This study was performed according to the declaration of Helsinki. RESULTS
All patients were clinically diagnosed as having MELAS, and an A-to-G transition mutation at nucleotide 3,243 (Cambridge number15) was detected in mtDNA in white blood cells or autopsied tissues by restriction enzyme analysis of mtDNA. In patient 1, mtDNA was detected in all of the autopsied organs, including cardiac muscle. Cardiac studies with echocardiography and stress myocardial scintigraphy with dipyridamole are shown in Table II. Cardiac catheterization studies indicated normal systolic function and normal coronary angiography. Moderate hypertrophy of the cardiac
muscles was found by M-mode echocardiography; decreases in E, integrated velocity for total E, and E/A ratio obtained from a pulsed Doppler examination showed decreased diastolic left ventricular function. In dipyridamole stress scintigraphy arterial insufficiency, that is, hypoperfusion in the early image and a “filling in” pattern in the late image, was found in the small vessels of patients 1,3, and 4. These cardiac studies indicated the earliest signs of cardiac failure resulting from hypertrophic cardiomyopathy and cardioangiopathy. Findings of SPECT images and those of coronary angiography of a representative case (patient 1) are shown in Figs. 1 and 2, respectively. Activities of the enzyme complexes in mitochondria from the skeletal muscles of four patients are shown in Table III. The enzymatic activities of complex I were markedly decreased in all patients (2 % to 27% of the mean control value). The enzymatic activities of complexes II and V in patients 1 and 2 were about twice as high as the normal values. The enzymatic activities of the other complexes were within the normal range. Immunoblot analysis of the subunits in patients 1 and 2 are shown in Fig. 3. The amounts of complex I subunits, particularly of subunit 75 kDa, were decreased in the mitochondria
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Fig. 1. Coronary angiography of patient 1. No stenosiswas observed in coronary artery.
Fig. 2. SPECT imagesin dipyridamole stressscintigraphy of patient 1. Hypoperfusion (arrow) in early image {A and 6) and a “filling-in” pattern in the late image (C and D) were found.
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Fig. 3. Western blot analysis of subunits of complexes I, II, III, IV, and V. C, Control mitochondria (15 pg protein); P1, patient 1 mitochondria (15 rg protein); P2, patient 2 mitochondria (15 g protein); I, complex 1 (figures show molecular masses (KDa) of detected subunits of complex I); ZZ,complex II (figure shows molecular mass of subunit); III, complex III (Core, cl, and ISP show core proteins, cytochrome cl and ironsulfur protein); ZV, complex IV (2, 4, 5, 6, and 7 show subunits 2, 4, 5, 6, and 7); V, complex V (LYand 0 show subunits (Yand (3).
Ill. Enzymatic activities in mitochondria from skeletal muscle
Table
---
-..__-
Patient Complex I II II-III III IV V Activities
are expressed
1
2
3
4
3 (2) 672 (212)
36 (25) 765 (241)
36 (25)
39 (27)
102 (70)
157 (108)
607 (83) 457 (75) 148 (202)
397 (54) 421 (67) 156 (214)
958 (156) 59 (81)
730 (119) 83 (114)
in nmol/min/mg
of mitochondrial
protein.
Figures in parentheses
from the patients. The amounts of complex II subunits were increased, which is consistent with the results of enzymatic activity measurement, The amounts of complexes III, IV, and V were normal or slightly decreased. Light microscopy of the endomyocardial specimen from patient 1 revealed disorganization of the myofibrillar architecture, interstitial fibrosis, and vacuolization of myocardial cells in elastica Masson staining. Modified Gomori’s trichrome staining of the endomyocardial specimen from patient 4 showed a red-purple deposit mainly around the nucleus similar in appearance to the ragged-red fibers in the skeletal muscle (Fig. 4). Electron microscopy revealed increased numbers and size of mitochondria, concen-
are percentages
Control (mean 2 SD) 146 317 145 731 613 73
+ tk t k ?I
59 64 59 319 276 22
of mean control
Range 82-246 216-350 42-243 411-1117 325-l 143 30-97
values
tric configuration of cristae, decreased myofibrils (Fig. 5), and swollen endothelial cells, resulting in narrowing of the capillary lumen. These findings in heart muscle by light and electron microscopy were similar to those found in skeletal muscle. DISCUSSION
Recent advances in molecular medicine have emphasized that mutations in mtDNA are of primary importance in the development of mitochondrial dysfunction, although the mitochondrial energytransducing system is also vulnerable to various environmental factors such as malnutrition, ischemia, hormonal disturbances,16 drugs,l’ and poisoning.lsy I9 It has previously been reported that hypertrophic
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Fig. 4. Modified Gomori’s trichrome staining of biopsied endomyocardial specimen from patient 4. Redpurple deposit around nucleus is similar to appearance of ragged-red fibers in skeletal muscle.
cardiomyopathy is associated with dysfunction of the mitochondrial electron-transfer system,2o-22 and deletion of mtDNA and several point mutations in mtDNA have been reported in patients with hypertrophic cardiomyopathy.23$ 24 These findings suggest that genetic disturbance of the mitochondrial energy-transducing system is an important factor in hypertrophy of cardiac muscle. In the present study abnormal mitochondria in endomyocardial specimens were observed morphologically. Mitochondria carry out the Krebs cycle and ,&oxidation pathway for fatty acids. These degenerative sequences essentially remove hydrogen from metabolic fuels with the release of carbon dioxide and transfer it via coenzymic carriers to the mitochondrial respiratory chain. The chain then transfers these reducing equivalents, eventually to react with molecular oxygen in the production of water. Reducing equivalents, NADH or succinate, are transferred via complex I, NADHubiquinone oxidoreductase, or via complex IT, succinate-ubiquinone oxidoreductase, respectively; to complex III, ubiquinol-cytochrome c oxidoreductase; and then to complex IV, cytochrome c oxidase, as shown in Fig. 6. These complexes, together with
complex V, adenosine triphosphatase, are responsible for the overall process of oxidative phosphorylation, i.e., energy production. In the present cases the activity of complex I was decreased and the activity of complex II was increased. Deterioration of complex I activity might induce this compensatory change. Because deterioration of complex I was observed in mitochondrial neuromyopathy6 and Parkinson’s disease,25 and because various agentslgp 26 and agingz7 also show detrimental effect to the activity of complex I, the role of complex I might be emphasized in the genesis of mitochondrial disease. Complex I is the largest of the respiratory chain complexes.and seems to be the most vulnerable to disturbance of membrane structural integrity. In addition, the vulnerability of mtDNA is expected to be much higher than the vulnerability of nuclear DNA.’ Because mtDNA encodes 7,3,2,1 and no subunits of complexes I, IV, V, IIT, and II, respectively, the effect of mutation of mtDNA on the vulnerability of complex I may be larger than that on other complexes. In the present study, biochemical analysis demonstrated markedly decreased activities of complex I in biopsied skeletal muscles. Immunochemical analysis
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Fig. 5. Electron micrograph showsA, Giant mitochondria (right); B, vacuolization of mitochondria fleftl in biopsied endomyocardial specimenfrom patient 1.
Complex I NADH
I.3 Cyt. a
02
% a3
Complex II
Complex Ii I
Complex IV
6. Schematicrepresentation of mitochondrial electron transfer system.FE, acid extractable Aavin; Fs, acid nonextractable flavin; nH Fe, nonhemeiron; Cyt, cytochrome; Cu, copper.
Fig.
also indicated
a decreased amount
of complex I sub-
units in cardiac muscles. An A-to-G transition at 3,243 in the tRNALeU(UUR) gene was observed in all four cases studied. This mu-
tation has been reported to cause an impairment of mtDNA-encoded protein synthesis,2” and this mutation results in severe impairment of 16s rRNA tranbiologic scription termination. 2g The molecular mechanism responsible for proliferation of mito-
chondria with mutated mtDNA remains obscure, however, and further investigations are required for clarification.
In addition to hypertrophic
cardiomy-
opathy, mtDNA mutations at 3,243 are often associated with the Wolff-Parkinson-White syndrome.24T 3o We also found mtDNA mutations in patients with conduction block.31 These results suggest that mtDNA mutations may be implicated as a cause of conduction disturbances.
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Sat0 et al.
Involvement of mtDNA mutation in angiopathy has rarely been reported. In evaluation of patients with stress scintigraphy with dipyridamole, three patients showed ischemia-like changes. Coronary angiography, however, did not demonstrate narrowing of the large vessels in these three patients. Therefore this arterial insufficiency might be the result of lesions in smal1 arterial vessels of the heart. Indeed, we observed marked increase in the number and size of mitochondria in endothelium and narrowed lumen of the arterioles. Ohama et a1.7observed similar findings in cerebral arteries of patients with MELAS, where a striking increase in the number of mitochondria in smooth muscle and endothelial cells occurred. On the basis of our results, we speculate that some patients with ischemic ST-T segment changes seen electrocardiographically but without narrowing of the large vessels angiographically might have angiopathy from mtDNA mutations. There are two or three copies of mtDNA in each mitochondrion, and several thousandmtDNA may exist in a ce11.32It is known that mtDNA with mutation is not distributed uniformly in the tissue33 and that tissue having mtDNA with mutation over a certain level might be expected to show deleterious function. These results present a challenge to the traditional treatment in those patients who have previously been considered to have arteriosclerosis. In addition, as seen in patient 3, hypoperfusion found on stress scintigraphy in a patient without cardiac hypertrophy can occur, indicating that angiopathy can be seen independently of cardiac hypertrophy. In conclusion, cardiomyopathy and/or angiopathy may be observed in patients with mtDNA mutation, and it is possible that other formal coronary artery disease such as arteriosclerosis may have an underlying mtDNA mutation. This possibility warrants further mtDNA study in coronary artery disease. We thank M. Bodman. our department language consultant, for reading the previous draft and making suggestions on language and style. REFERENCES
Linnane AW, Marzuki S, Ozawa T, Tanaka M. Mitochondrial DNA mutations as an important contributor to aging and degenerative diseases. Lancet 1989;1:642-5. Pavlakis SG, Phillips PC, DiMauro S, De Vito DC, Rowland LP. Mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes: a distinctive clinical syndrome. Ann Neurol 1984;16:481-8. Goto Y, Nonaka I, Horai S. A mutation in the tRNALeu(U”R) gene associated with the MELAS subgroup of mitochondrial encepholomyopathies. Nature 1990;348:651-3. Kobayashi Y, Momoi MY, Tominaga K, Momoi T, Nihei K, Yanagisawa M, Kagawa Y, Ohta S. A point mutation in the mitochondrial tRNALeu(UUR) gene in MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). Biochem Biophys Res Commun 1996;173:816-22.
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5. Tanaka M, Ino H, Ohno K, Ohbayashi T, Ikebe S, Sano T, Ichiki T, Kobayashi M, Wada Y, Ozawa T. Mitochondrial DNA mutations in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Biochem Biophys Res Commun 1991;174:861-8. 6. Ichiki T, Tanaka M, Nishikimi M, Suzuki H, Ozawa T, Kobayashi M, Wada Y. Deficiency of subunits of complex 1 and mitochondrial encephalomyopathy. Ann Neurol 1988;23:28794. 7. Ohama E, Ohara S, Ikuta F, Tanaka K, .Nishizawa M, Miyatake T. Mitochondrial angiopathy in cerebral blood vessels of mitochondrial encephalomyopathy. Acta Neuropathol 1987;74:226-33. 8. A.H.A. Committee Report. A reporting system on patients evaluated for coronary artery disease. Circulation 1975;51:540. 9. Ota Y, Tanaka M, Sato W, Ohno K, Yamamoto T, Maehara M, Negoro T, Watanabe K, Awaya S, Ozawa T. Detection of platelet mitochondrial DNA deletions in Kearns-Sayre syndrome. Invest Ophthalmol Vis Sci 1991;32:2667-75 10. Bookelman H, Trijbels JMF, Sengers RCA, Janssen AJM. Measurement of cytochromee in human skeletal muscle mitochondria, isolated from fresh and frozen stored muscle specimens. Biochem Med 1978;19:366-73. 11. Yoneda M, Tanaka M, Nishikimi M, Suzuki H, Tanaka M, Nishizawa M, Atsumi T, Ohama E, Horai S, Ikuta F, Miyatake T, Ozawa T. Pleiotropic molecular defects in energy-transducing complexes in mitochondrial encephalomyopathy (MELAS). J Neurol Sci 1989;92:143-58. 12. Tanaka M, Miyabayashi S, Nishikimi M, Suzuki H, Shimomura Y, Ito K, Narisawa K, Tada K, Ozawa T. Extensive defects of mitochondrial electron-transfer chain in muscular cytochrome c oxidase deficiency. Pediatr Res 1988;24:447-54. 13. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH. Provenzano MD. Fuiimoto EK. Goeke NM. Olson BJ. Klenk DC. Measurement of protein using bicinchbninic acid: Anal Biochem 1985;150:76-85. 14. Kobayashi Y, Miyabayashi S, Takada G, Narisawa K, Tada K, Yamamoto TY. Ultrastructural study of the childhood mitochondrial myopathic syndrome associated with lactic acidosis. Eur J Pediatr 1982;139:25-30. 15. Anderson S, Bankier AT, Barrel1 BG, de Bruijn MHL, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJH, Staden R, Young IG. Sequence and organization of human mitochondrial genome. Nature 1981; 290:457-65. 16. Sugiyama S, Kato T, Ozawa T, Yagi K. Deterioration of mitochondrial function in heart muscles of rats with hypothyroidism. J Clin Biochem Nutr 1991:11:199-204. 17. Ogawa Y, Kondo T, Sugiyama S, Ogawa K, Satake T, Ozawa T. Role of phosphotipase in the genesis of doxorubicininduced cardiomyopathy in rats. Cancer Res 1987;47: 1239-43. 18. Kato T, Sugiyama S, Hanaki Y, Fukushima A, Akiyama N, Ito T, Ozawa T. Role of acetylchohne in pyridostigmine-induced myocardial injury: possible involvement of parasympathetic nervous system in the genesis of cardiomyopathy. Arch TOXicol 1989;63:137-43. 19. Sugiyama S, Takasawa M, Hayakawa M, Esumi H, Ozawa T. Detrimental effects of 2-amino-1-methyl-6-phenylimidaxo[4,5-blpyridine, a mutagenic agent, on mitochondrial respiration among various rat tissues. Biochem Mol Biol Int 1993; 30:797-805. 20. Papadimitriou A, Neustein HB, DiMauro S, Stanton R, Bresolin N. Histiocytoid cardiomyopathy of infancy: deficiency of reducible cytochrome b in heart mitochondria. Pediatr Res 1984;18:1023-8. 21. Zeviani M, VanDyke DH, Servidei S, Bauserman SC, Bonilla E, Beaumont ET, Sharda J, VanderLaan K, DiMauro S. Myopathy and fatal cardiopathy due to cytochrome c oxidase deficiency. Arch Neurol 1986;43:1198-202. 22. Smeitink JAM, Sengers RCA, Trijbels JMF, Ruitenbeek W,
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Da&Is 0, Stadbouders AM, Kock-Jansen MJH. Fatal cardiomyopathy associated with cataract and mitochondrial myopathy. Eur J Pediatr 1989;148:656-9. 23. Hattori K, Ogawa T, Kondo T, Mochizuki M, Tanaka M, Sugiyama S, Ito T, Satake T, Ozawa T. Cardiomyopathy with mitochondrial DNA mutations. AM HEART J 1991;122:866-9. 24. Obayashi T, Hattori K, Sugiyama S, Tanaka M, Itoyama S, Deguchi H, Kawamura K, Koga Y, Toshima H, Takeda N, Nagano M, Ito T, and Ozawa T. Point mutations in mitochondrial DNA in patients with hypertrophic cardiomyopathy. AM HEART
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.%I!:,
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segregate with the tRNALeu(liUR) mutation associated with mitochandrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes. Mol Cell Biol 1992;12:480-90. 29. Hess JF, Parisi MA, Bennett JL, Clayton DA. Impairment of mitochondrial transcription termination by a point mutation associated with the MELAS subgroup of mitochondrial encephalomyopathies. Nature 1991;351:236-9. 30. Zeviani M, Gellera C, Antozzi C, Rimoldi M, Morandi I,, VilIani F. Tiranti V. DiDonato S. Maternallv inherited mvonathv and cardiomyopathy: association with mutation in mitochondrial DNA tRNAL”U’U”R’. Lancet 1991;338:143-7. 31. Sato W, Tanaka M, Sugiyama S, Hattori K, Ito T, Kawaguchi H, Onozuka H, Yasuda H, Ito K, Takada G, Ozawa T. Deletion of mitochondriai DNA in a patient with conduction block. I
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32. Robin ED, W ong R. Mitochondrial DNA molecules and virtual number of mitochondria per ceII in mammalian cells. J Cell Physiol 1988;136:50’7-13. 33. Ozawa T, Tanaka M, Sato W, Ohno K, Sugiyama S, Yoneda M, Yamamoto T, Hattori K, Ikebe S, Tashiro M, Sahashi K. Mitochondrial DNA mutations as an etiology of human degenerative diseases. In: Ozawa T, Kim CH. eds. Bioenergetits. New York: Plenum Press, 1990:413-27.