Mitochondrial DNA deletions in dilated cardiomyopathy: A clinical study employing endomyocardial sampling

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ANNE M. REMES, MD, PHD, ILMO 11 11ASSINEN, MD, PIED, MARKKU J . IKAHEIMO, MD, PIED, RUTTA HERVA, MD, PHD, JORMA IIIRVONEN, MD, PHD, KEIJO J . PEUHKURINEN, MD, PH Oulu, Finland

Objectives. The aim of this study was to assess the occurrence of the two most commonly encountered aflauchondrial DNA (mIDNA) deletions in the hearts of patients with idiopathic dilated cardlomyepathy . Background. The mutation frequency of natDNA is high, and sporadic cases. of cardiomyopathles associated with mIDNA delelions ho* ,.. hen itscribed. Reports of increases in mIDNA deletions with advancing age also exist . mi%wh. %e studied 15 consecutive patients with typical signs of idiopathic dilated cardiomyopathy, without a family history, together with 10i control hearts obtained at autopsy from patients who died of noucardiac causes . The patients underwent both right and left heart catheterization, dun ing which endomyoc2rdial biopsy samples were taken. The mtDNA in these samples and in the control hearts was analyzed by the polymerase chain reaction technique for the occurrence and proportion of 5- and 7 .40lobase (kb) deletions (Cambridge sequence map positions from nucleotides 849P to 134"47 and 8637 to 16084, respectively) . Results . The 5-kb mfDNA deletion was observed in the hearts

of all of the patients with idiopathic dilated cardiomyopathy, accounting for 0.32 :k 0.05% (mean ± SEM) of the total miDNA. The 7,4-kb deletion was found in 7 of the 15 patients with idiopathic dilated cardiomyepathy and comprised 0 .28 :t 0 .08% of the total. The 5- and 7 .4-kb deletions were detected in 12 and 9 control hearts, respectively, quantitatively similar to the patients with idiopathic dilated cardiomyopathy . A sigmoidal age dependency of the mIDNA deletions was found both in the patients with cardiomyopathy and in the control hearts, but after elimination of the confounding age variable, there was no difference between these groups . Conclusions . Because of the similarity of the age-dependent increase in the frequency of antDNA deletions in cardiomyopethic and control hearts, the deletions have no causal relation with idiopathic dilated cardiomyopathy . The present results confirm the notion of an increase in anIDNA deletions with advancing age and show that endomyocardial tissue sampling is a feasible method for detecting mtDNA defects in affected hearts . U A in Coll Cardiol 1994,23:935-42)

Idiopathic dilated cardiomyopathy is probably mullifactorial in origin (1,2), and the possibility of a viral etiology is supported by increased titers of antibodies to Coxsackie virus or the occurrence of enterovirus RNA in the cardiac tissue of some patients (3-5), although the enterovirus hypothesis has been challenged recently (6) . The other major hypothesis, involving autoimmunity-mediated damage to myocytes, is supported by the presence of autoantibodies to apparently normal or abnormal cardiac tissue (7-11) . Defects in natural killer (12) and suppressor lymphocyte activity (13,14) and myocyte-specific cytotoxic T-cell responses (15) have also been detected, arguing for an immunologic etiology . Predisposition to the disease may be determined by the

major histocompatibility complex genes (16,17), and Mar-

From the Departments of Medical Biochemistry, Internal Medicine, Pathology and Forensic Medicine, University of Oulu and Oulu University Central Hospital, Oulu, Finland . This study was supported by the Sigrid Juselius Foundation, the Finnish Foundation for Cardiovascular Research and the Medical Council of the Academy of Finland, Helsinki . Manuscript received June 15,1993 ; revised manuscript received October 25, 1993, accepted November 9, 1993 . Address for co : Dr . KeUo J . Peuhkurinen, Department of Internal Medicine, Division of Cardiology, Oulu University Central Hospital, Kajaimintir. 50, SF-90220 Oulu, Finland . 01994 by the American College of Cardiology

gossian et al . (18) have recently reported a reduction in the myocardial regulatory light chain content of myosin (LC2), related to the presence of an active protease . The proteasemediated cleavage of LC2 in myosin would, according to their hypothesis, lead to changes in myosin structure and function, with signs of idiopathic dilated cardiomyopathy . Cases of familial aggregation emphasize the potential role of genetic factors in the pathogenesis of idiopathic dilated cardiomyopathy (19-22), and some cases of isolated cardiomyopathy with deletions or point mutations in mitochondrial DNA (mtDNA) have been described recently (23-27) . Mitochondrial DNA is a small (16 .5-kilobase [kb]), circular, double-stranded molecule that contains 13 genes for peptides of the mitochondrial respiratory chain, 2 for mitochondrial ribosomal RNAs and genes for all 22 transfer RNAs required for mitochondrial protein synthesis (Fig . 1) (28) . Previous research has indicated impaired mitochondrial function in the failing human heart (29,30), but this has not been attributed to defects in mtDNA . Most idiopathic dilated cardiomyopathy seems to he nonfamilial . However, and as with most mtDNA deletions, there is no obvious maternal inheritance . We therefore set

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ATPase 8

Figure 1 . Gene map of the mitochondrial DNA showing the location and extent of the 5- and 7 .4-kilobase (kb) deletions. PCRI, PCR2 = regions amplified by the polymerise chain reaction for the detection of the 5- and 7 .4-kb deletions, respectively ; ND 1-5 = nicotinamide adenine dimucleotide subunit genes (complex 1) ; Cyto B = cytochrome b ; CO = cytochrome oxidase (complex IV) ; ATPase = adenosine triphosphate synthetase (complex V) ; f-Met = fMet peptide; D-loop = displacement loop. Amino acids indicate the corresponding amino acid transfer RNA genes .

out to study the occurrence of the two most common

mitochondrial defe7ts, the 5- and 7 .4-kb mtDNA deletions (31,32), in the myocardium of a prospective series of 15 patients with idiopathic dilated cardiomyopathy without any family history of the disease . Tissue samples were obtained by endomyocardial biopsy, a technique that we recently used succesdk$r to diagnose myocardial mtDNA deletion in a patient with a third-degree atrioventricular block and Kcams-Sayre syndrome (33) . Because it has been reported that the frequency of mtDNA deletions increases with advancing age (32,34-38), an additional i6 hearts obtained at autopsy from subjects who died of noncardiac causes were also studied .

Methods Patients ad dbpodk procedures . These series comprised 15 patients with symptoms of heart failure and typical echocardiographic features of idiopathic dilated cardiomyopathy, none of whom had a positive family history of cardiomyopathy . All of them underwent cardiac catheterization . Intracardiac pressures were monitored during rightsided catheterization, and cardiac output was determined by the Fick principle . In addition, 10 endomyocardial biopsy samples were obtained from the right ventricular septum or apex in each patient, of which 5 were used to analyze tissue

JACC Vol . 23, No . 4 March 15, M : 9 35-42

morphology, and 5 were frozen in liquid nitrogen for subsequent DNA analysis . Left ventricular pressures were measured during left-sided catheterization, and ventriculography and coronary angiography were performed . All patients n=t be diagno-stic critteena put. 'fonvard for idiopathic dilated cardiomyopathy by Manolio et al . (2), and their clinical characteristics, medication and echocardiographic and catheterization findings are shown in Table 1 . Most patients were in New York Heart Association functional class 11 to 111 . Mean (±SEM) left ventricular enddiastolic diameter was 73 ± 2 mm, and mean ejection fraction was 30 ± 3% by M-mode echocardiography . Only one patient had signs of coronary artery disease (<50% diameter stenosis in the left anterior descending coronary artery), and none had histopathologic signs of active myocarditis . Control samples were obtained at autopsy from the hearts of 16 subjects who had died of noncardiac causes (Table 2) . There were no macroscopic or microscopic signs of cardiac pathology in the control hearts . The research was approved by the local ethical committee of the Medical Faculty of Oulu University, and all patients with idiopathic dilated cardiomyopathy gave informed consent before the catheterization examination . DNA analysis . Total DNA was isolated from 10 to 30 mg of endomyocardial biopsy or autopsy sample by sodium dodecyl sulfate-proteinase K digestion at 42°C, followed by phenol/chloroformfisoamyl alcohol extraction (39), and the mtDNA deletions were examined by digesting 2 /Ag of the total DNA with Pvu II restriction enzyme, which has only one site for cutting mtDNA and thus linearizes the mtDNA molecule . The DNA fragments were separated electrophoretically and transferred to nitrocellulose filters that were hybridized with phosphorus-32-radiolabeled whole human mtDNA and autoradiographed overnight at 72°C using Kodak XAR film with an intensifying screen . To detect small amounts of the 5-kb deletion (Cambridge sequence map position from nucleotide 8469 to 13447) (Fig . 1), the specific proportion of mtDNA was amplified by the polymerase chain reaction technique (40) . The primers used for polymerase chain reaction amplification were positioned at nucleotides 8282 to 8305 and 13851 to 13832 (593 base-pair product) . The polymerise chain reaction was carried out in a 100-pJ reaction volume containing 200 JLmolffiter of each deoxynucleotide triphosphate (deoxyadenosine triphosphate deoxycytosine triphosphate, deoxyguanosine triphosphate and deoxythymidine triphosphate), 50 mmollliter of potassium chloride, 1 .5 mmol/liter magnesium chloride, 0.01% (wt/vol) gelatin, 100 pmol of each primer and 2 .5 IU of Taq polymerase (Promega) . Twenty-five nanograms of the total DNA was used for each amplification . There were 30 polymerase chain reaction cycles of denaturing at 94°C for 30 s, annealing at 56°C for 1 min and extension at 72°C for 1 min, except that the first cycle had 2 min of denaturation . The generated mtDNA fragments were visualized on 1% agarose gel with ethidium bromide .



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Table 1 . Clinical Characteristics of Patients With Dilated Cardiomyopathy Pt no .

1

Age (yr)/gender Duration of symptoms (yr)

2

25/M

3

45/M

4

5

46/M

481M 4

Ill

111

H

7 52/M 1

3

9

10 1I

4 II

113

95

63

66

72

78

66

83 26

53 33

49 49

66 17

61 38

51 44

54

44

30

34

74 63 31

8 9

36 23

27

19

26

ND

ND

8

PCWP (mm Hg)

23 215

ND

0 .37

12 3 .53

20

PVR (Wood res units)

13 3 .4

4 3 .2

4 .5

6 .7

4 .88

NYHA functional class

461'M 3

6

45/M 2

II

52/F

53/M

2 11

5 11

33

10 56/M

11 601M

12 601M

13

14

61/61

61/M 5

3

1

11

11

1 II

74 63

79 71

78 71

30

27

19

18

22

16

13

23 1 .7

33 29

3 31

15 63,tM 1

52 - 3 3 .1 ± 0 .6

I

Ill

76

72

64

66

66 51

61 ± 3

20

44

30 ± 3

22

22 .1 ± 2 .5

CAD Echocardiographic finding LVEDD (mm) LVESD (mm) EF (0170 Finding at catheterization LVEDP (mm Hg)

CO (liters/min) Medication

5 .3

Digitalis Diuretic agents

+ -

ACE inhibitors

+

Long-acting nitrates Beta-blockers

0.98 4 .05

IJ 3.6

ND ND

4 .3

11 4.64

4 .7

3 .08 2 .91

19

ND

29

21 3 .0

39 3 .92

ND ND

31 5 .2

2.6

4 .58

3 .74

4 .3

73 ± 2

20 ± 3 2 .6 a 0 .4 4 .3 ± 0 .3

+ + +

Anticoagulant agents

+ I.

Antiarrhythmic agents

+ -

ACE = angiotensin-converting enzyme ; CAD = coronary artery disease ; CO = cardiac output ; EF = ejection fraction ; F = female ; LVEDD = left ventricular end-diastolic diameter; LVEDP = left ventricular end-diastolic pressure ; LVESD = left ventricular end-systolic diameter ; M = male ; ND not detectable ; NYHA = New York Heart Association -, PCWP = pulmonary capillary wedge pressure ; Pt = patient ; PVR = pulmonary v.;culgr resistance ; res = resistance .

The 7.4-kb deletion (nucleotide 8637 to 16084) (Fig, 1) was detected by amplifying total DNA with primers positioned at nucleotides 8150 to 8166 and 16159 to 16142 (574 base-pair fragment) . The conditions for polymerase chain

2 . Frequency of Mitochondrial DNA Deletions and Proportion of Total Mitochondrial DNA in Control Subjects at Autopsy Table

Case No.

Age (yr)/ Gender

5-kb Deletion (To of total

7 .4-kb Deletion (% of total

mtDNA)

mtDNA)

ND

ND ND ND

Cause of Death

1 2

21 wk*/M 38 wk*/F

3

21M

ND ND

4

12/M

ND

ND

Suicide

5

30/F

0 .124

0 .089

Suicide

6

321M

0 .112

0 .031

7 8

32/F 35/F

0 .232 0 .312

ND ND

Suicide Alcohol intoxication

9

38/M

0 .431

0 .286

Accident

10

411M

0 .400

0 .217

II

0 .306

ND

Suicide Accident

12

43/M 44/M 441M

0 .421 0.586

0 .395 0 .537

Suicide

13 14

48/M

Suicide

651M 77/M

0.324 0.378

0 .353

15

0 .529

0.650

0 .453

Rupture of iliac artery Accident

16 Mean tSEM

0.36 ±0.05

13-Trisomy Caudal anomaly Accident

Suicide

Subarachnoid hemorrhage

0 .32 ±0.06

* Fetal age . kb = kilobase; other abbreviations as in Table 1 .

reaction amplification were the same as those described for the 5-kb deletion, except that the annealing temperature was 52°C . The amplified products were identified by direct sequencing with the same oligonucleotide primers as those used for amplification (41) . The deleted mtDNA was quantified by amplifying specific regions of the mtDNA in the presence of 5 juCi of sulfur35-dATP . As an internal control the mtDNA of each sample was also amplified from a region where deletions have rarely been described (31), using oligonucleotide primers from nucleotides 800 to 825 and 1350 to 1326, which generate a mtDNA fragment about equal in size to the amplified 5- and 7 .4-kb deleted regions . Thus, quantitative data were obtained despite the size dependence of the efficiency of the polymerase chain reaction technique . Ten nanograms of total DNA was used for one polymerase chain reaction . The conditions for the amplification were the same as those described for the 5-kb deletion, except that the annealing temperature was 66°C . The ratio of deleted to total mtDNA was ascertained by electrophoresis through a 5% nondenaturing polyacrylamide gel with appropriate dilutions . The gels were dried and autoradiographed at -72°C for 48 h using Kodak XAR film with an intensifying screen, and the autoradiographs were quantified with a Bioimage scamicr (Millipore) . Several dilutions of total DNA were used for the polymerase chain reaction to optimize the linear phase of the exponential amplification, and 10 and 25 ng of total DNA were used to amplify the total and deleted mtDNA, respectively, these amounts having been selected after optimization in trials with several dilutions .



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MMopwMaW, The endomyocardial biopsy samples were studied with both light and electron microscopy . Consecutive cryostat sections were stained with hematoxylineosin, periodic acid-Schiff, Gomori trichrome, Herovici, Sudan black and Berlin blue stains . The specimens for electron microscopy were fixed in 4% formaldehyde/l% glutaraldehyde buffered to pH 7 .4 with phosphate buffer and were postfixed in osmium tetroxide and embedded in Epon LX-112. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined in a Philips LS electron microscope . ShOWK Logistic regression analysis was used to allow for the influence of age when testing the connection between mitochondria) DNA deletions and idiopathic dilated c6rdiomyopathy, using the SPSS (release 5 .0 for MS Windows, SPSS, Inc.) program package (42) . Nonlinear curve fitting was performed with the Marquardt algorithm using the Statgraphics version 2 .6 (Manugistics, Inc .) and Sigmaplot version 4 .0 (Jandel Scientific) program packages .

Table 3. Frequency of Mitochondrial DNA Deletions and Proportion of Total Mitochondrial DNA in Patients With Idiopathic Dilated Cardiomyopathy

Pt No .

Age (yr); Gender

5-kb Deletion (% of total mtDNA)

7 .4-kb Deletion (% of total mtDNA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

251M 451M 45/M 461M 46/F 48/ni 52/m 52/M 531M 56/M 601M 601M 61161 61/M 63/M

0 .024 0 .037 0 .354 0 .048 0 .341 0 .491 0.278 0.171 0 .520 0.300 0.611 0.708 0.393 0.335 0.128 0 .32 ±0405

ND ND ND ND ND 0.072 0.237 ND 0.230 TIV 0.102 0.457 ND 0.668 ND 0.28 ±0.08

HEM

mtDNA = mitochondrial DNA, other abbreviations as in Tables I and 2.

Results After Southern blot hybridization of the endomyocardial DNA failed to detect mtDNA deletions in the patients with idiopathic dilated cardiomyopathy, the biopsy samples were screened with the polymerase chain reaction for two of the most common mtDNA defects : the 5-kb deletion (Cambridge sequence map position 8469 to 13447, coding four subunits of complex 1, one of complex IV and one of complex V of the mitochondrial respiratory chain and five transfer RNAs) and the 7 .4-kb deletion (8637 to 16084, coding five subunits of complex I, one of complex III, one of complex IV and one of complex V of the mitochondrial respiratory chain and eight transfer RNAs). The 5-kb deletion was observed in the hearts of all of the patients with clinically typical idiopathic dilated cardiomyopathy, the proportion of the mtDNA deleted varying from 0 .02% to 0 .71% (0 .32 ± 0.05% [mean ± SEM]) (Table 3) . The 7 .4-kb deletion was found in only 7 of the 15 patients and varied from 0.07% to 0 .67% (0 .28 ± 0.08% [mean ± SEM]) . The 5- and 7.4-kb deletions were detected in 12 and 9 control hearts, respectively, but it should be noted that they were not found in the four youngest hearts and that all of the control hearts with the 5 kb-deletions were from subjects >30 years old (Table 2) . The 5- and 7 .4-kb deletions increased sigmoidally in extent with advancing age in both groups (p < 0 .0001) (Fig . 2). Logistic regression was used to test the contribution of the mtDNA deletions to the probability of cardiomyopathy . If the dependent variable (y) is given a value of 1 fur cardiomyopathy and 0 for health in the model, then the odds can be calculated from the equation (P(Y=l))I(P(Y=-')) = exlKbo + b l x l + bkXA where the b, . . . k terms are the risk coefficients for the independent variables x, . . . kWhen deletion frequency and age and gender were included in the model, the results of the statistics are as shown

in Table 4 . Classification using the model containing the sum of the 5- and 7.4-kb deletions was correct in 77 .4% of cases, with a specificity of 81 .3% and a sensitivity of 73 .3% . It can be deduced from Table 4 that there was a statistically significant contribution of age to the occurrence of the idiopathic cardiomyopathy . The risk coefficient of the deletion frequency was negative, with a large standard error. Because the concept of a protective effect of a deficiency in mtDNA can be abandoned, this can be interpreted to indicate that the mtDNA deletions do not contribute to the development of idiopathic cardiomyopathy . There was no statistically significant contribution of gender to the occurrence of the cardiomyopathy . Performance of the polymerase chain reaction system for detecting the two mtDNA deletions in the endomyocardial biopsy samples is demonstrated in Figure 3 . Light microscopy of the biopsy samples from the patients with idiopathic dilated cardiomyopathy generally showed some fibrosis but no inflammatory cells or increased iron deposits, whereas electron microscopy revealed degenerative changes, loss of myofilaments and large amounts of lipofuscin. Three patients (Patients 6, 12 and 13, Table 3) had accumulations of lipid droplets next to abnormally large mitochondria (i.e ., >1 pm in diameter) . Some of these mitochondria showed structural variation, concentric cristae and osmiophilic inclusions (Fig . 4) . Ragged red fibers, as encountered in the skeletal muscles of patients with mitochondrial myopathy (33,43-45), were not present, however .

Discussion Occurrence of mtDNA deletions in heart muscle. The results show !hat the 5- and 7 .4-kb deletions in mtDNA are



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Table 4 . Logistic Regression Using Idiopathic Cardiomyopathy as the Dependent Variable Risk Coefficient

SE

p Value

5-kb Deletion frequency

-4 .929

3 .068

0 .1051

Age

0.112 -0.047

0 .045

0 .0132

0.655

0 .945

-5 .326 0.156 0.726

2 .433 0 .0597 1 .450

0 .0296 0 .0087 0 .617

Model

Gender Sum of 5- and 7.4-kb deletion frequencies

Age Gender kb = kilobase,

disease (from 0 .017% to 0 .16%) . Two of these patients had idiopathic dilated cardiom)opathy (46) . The proportion of the 5- and 7 .4-kb deletions determined 1 .4

here in patients with idiopathic dilated cardiomyopathy and in control hearts was higher, but it should be noted that

1 .2

amplifying DNA with the polymerase chain reaction technique is highiy dependent on external circumstances, and the

1 .0

discrepancies in the percents obtained in various surveys

Figure 3 . Detection of deleted mtDNA in the hearts of patients with idiopathic dilated cardiomyopathy and in control subjects by the polymerase chain reaction . Top, Electrophoretogram showing 0 .6kilobase (kb) amplified mtDNA fragments, indicating a 5-kb deletion . Lane I = control subject without the deletion ; lanes 2 and 3 = patients with idiopathic dilated cardiomyopathy . The fragment that

0 .0

shows normal mtDNA fails to amplify under the conditions used . Bottom, Electrophoretogram demonstrating 0 .6-kb amplified mtDNA fragments corresponding to the 7 .4-kb deletion . Lane I = control subject with the deletion ; lanes 2 and 3 = patients with idiopathic dilated cardiomyopathy patients with and without the deletion, respectively . The fragment representing normal mtDNA fails to amplify under the conditions used .

Figure 2. Plots showing the age dependence of the mitochondrial 5-kb DNA deletion (top) and the sum of the 5- and 7 .4-kilobase (kb) deletions (bottom) in the endomyocardium of patients with idiopathic dilated cardiomyopathy (solid circles) and in control hearts (open circles) . Curves = nonlinear fits of the logistic equation y = Ax exp[B(x - C)]/{I + exp[B(x - Q]' to the data points ; dotted, dashed and solid curves = fits to the control, idiopathic dilated cardiomyopathy and combined data, respectively .

very common in patients with idiopathic dilated cardiomyopathy, but they seem to occur at approximately the same frequency in an age-matched control group . Cortopassi and Arnheim (36), studying human tissue samples collected at autopsy, found that normal adult heart and brain tissues contain both normal-sized and deleted mtDNA and are therefore heteroplasmic . They estimated that the 5-kb deletion accounted for -0 .1% of the total mtDNA . CorralDebrinski et al . (46) reported a frequency of 0 .0035% for the 5-kb mtDNA deletion in normal hearts, whereas higher amounts of this deletion were found in ischemic hearts (from 0 .02% to 0 .85%) and in 10 patients with other types of heart

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REMES ET AL . MITOCHONDRIAL DNA DELETIONS IN ENDOMYOCARDIUM

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Rpre 4. Electron micrograph of endomyocardial heart muscle from Patient 6 . Several abnormal mitochondria with concentric cristae are present . Lead citrate, uranyl acetate staining. x 12,W9 reduced by 35% .

may at least partly be explained by methodologic differences . Apt dqxudellcy. Sugiyama et al . (38) reported that the proportion of the 7 .4-kb deletion in mtDNA from cadavers of patients who died of noncardiac causes varies considerably (from 0 .025% to 10%), increasing exponentially with age . In fact, it has been suggested that the increase in the frequency of mtDNA deletions with advancing age may be one factor contributing to presbycardia (32,34-37). The age-dependence of the deletions was also seen in this series because neither of the two fetuses who died during the intrauterine period nor the two children in the control group had mtDN A deletions in their hearts . However, the 7 .4-kb deletion was not detected in all of the elderly control subjects or patients with idiopathic dilated cardiomyopathy (Table 3) . The general pattern, nevertheless, was for both mtDNA deletions to increase sigruoidally with advancing age (Fig . 2) . It has been shown that mtDNA evolves 5 to 10 times faster than single-copy nuclear DNA and that this may be due, at least in part, to an elevated rate of mutation in mtDNA . The high mutation rate of mtDNA probably results from several factors, such as lack of protective histones and adequate repair mechanisms, continuous exposure to free radicals produced in the mitochondrial respiratory chain and lligh turnover amd replication rate of mtDNA also in nondividing tissues (47,48) . It has been suggested that damage to mtDNA results in the accumulation of respiration-deficient cardiomyocytes in advancing age and that this imposes a cardiac limit on a person's life span (48) . It has also been suggested that changes in mitochondrial function are involved in the characteristic form of programmed cell death called apoptosis, which occurs in embryogenesis, tissue atrophy and tumor regression (49) . In this phenomenon the role of mtDNA remains controversial because during apoptosis, at least

some regions of mtDNA are relatively resistant to degradation compared with nuclear DNA (50). Thm specificity and degree of hetereplasmy. The degree of heteroplasmy (i .e ., deleted mtDNA relative to total mtDNA) was found to be low in patients with idiopathic dilated cardiomyopathy, comparable to the control hearts . Suomalainen et al . (27) described a woman and her son who both died of idiopathic dilated cardiomyopathy and were shown by Southern blot. hybridization and polymerase chain reaction analysis to have multiple large deletions in mtDNA, so that it was estimated that in the son the mtDNA population with deletions of different sizes comprised -50 of the total nnDNA (27). It thus seems that these two patients belong to a small subgroup with the hereditary form of idiopathic dilated cardiomyopathy wits marked defects in mtDNA, as also pointed out by these investigators . The Kearns-Sayre syndrome belongs to a category of mitochondrial myopathies with defects in mtDNA observed in several tissues, including heart muscle (44) . The deletions are heteroplasmic, and some tissues may be totally free of mtDNA defects, emphasizing the need for elective sampling of the target tissue in diagnostic testing (33) . Conversely, the proportion of the mtDNA deleted in the tissues involved ranges from 20% to 90% (44,45) and is thus much higher than that observed here in either the patients with idiopathic dilated cardiomyopathy or the control hearts . Despite this, the cardiac conduction system is usually selectively affected in the Kearns-Sayre syndrome (51-53), and only a few patients with signs of congestive cardiomyopathy have been described (54,55) . This raises the possibility that the KearnsSayre syndrome may involve enrichment of the cells of the conduction system, with mitochondria having mtDNA deletions, or that the cardiomyocytes in general are much more resistant to mtDNA defects than the cells of the conduction system .



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topatholuggy. The ultrastructural findings of the end myocardial biopsy samples from the patients with idiopathic dilated cardiomyopathy (i .e ., degeneration in myocardial cells and increased fibrosis) resembled those described by Schaper et al . (56) . Although ragged red fibers (accumulations of abnormal mitochondria) are often found in muscle tissue with large-scale mtDNA deletions or point mutations in mitochondrial transfer RNA (33), none of our patients with idiopathic dilated cardiomyopathy had any . Only some concentric cristae, also typical of the Kearns-Sayre syF1drome, were found in the mitochondria of three patients with an abundance of fat in the muscle fibers . Two of these three patients also had large amounts of deletions (Patients 6 and 12, Table 3), but the significance of this remains unclear . Significance of mIDNA deletions in other diseases . Many investigations have shown that in most cases where ocular myopathy is a key feature (Kearns-Sayre syndrome, chronic progressive external ophthalmoplegia), a high proportion of the total population of skeletal muscle nitDNA molecules house large-scale deletions (31,43) . Major deletions live also been found in cases of Pearson's marrow/pancreas syndrome (57) . Some isolated cases of myotonic dystrophy and dilated cardiomyopathy (23) associated with mtDNA deletions have been described . However, our study and another prospective analysis of 20 patients with typical myotonic dystrophy (58) showed that mtDNA deletions dnoyt play any significant role in the pathogenesis of these diseases . The same is true for X-linked cardioskeletal myopathy with neutropenia or Barth syndrome, where mitochondria also show abnormal cristae but where the genetic defect is located at Xq28 (59) . Conclusions . We showed that the 5- and 7 .4-kb mtDNA deletions, two of the most common, occur at the same frequency with a low percent of heteroplasmy in the heart muscles of patients with idiopathic dilated cardiomyopathy and in those dying of noncardiac causes . This supports the idea that mtDNA deletions, at least those studied here, do not play a major role in the etiopathogenesis of idiopathic dilated cardiomyopathy . They were not observed in the youngest control subjects, and they increased in amount with age, in accordance with earlier findings (33-35,37) . The results also confirm. a recent observation of ours that endomyocardial tissue sampling for analysis of mtDNA by the polymerase chain reaction technique is a feasible method for detecting mtDNA defects in affected heart muscle (33) . We thank Ms . Kaisu Korhonen and Ms . Anja Roukala for their expert technical assistance .

References 1 . Bender JR . Idiopathic dilated cardiomyopathy . An immunologic, genetic, or infectious disease, or all of the above? Circulation 1991 ;83:704-6 . 2. Manolio TA, Baughman KL, Rodebeffer R, et al . Prevalence and etiology of idiopathic dilated cardiomyopathy (summary of a National Heart, Lung, and Blood Institute workshop) . Am J Cardiol 1992 ;69 :1458-66.

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3 . Abelmana WH, Lorell Bit . The challenge of cardiomyopat .hy . J Am Colt Cardiol 1989;13:1219-39, 4 . Bowles NE, Richardson P1, Olsen GJ, Archard LC . Detection of Coxackie B virus-specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy . Lancet 1986 ;1 : 1120-3 . 5 . Bowles NE. Rose ML . Taylor P . et al . End-stage dilated cardiomyopathy . Persistence of enterovirus RNA in myocardium at cardiac transplantation and lack of immune response . Circulation 1989 :80:1128-36 . 6 . Keeling PJ, Jeffery S, Caforio ALP, et al . Similar prevalence of enteroviral genome within the myocardium from patients with idiopathic dilated cardiomyopathy and controls by the polymerase chain reaction . Br Heart J 1992 ;68 :554-9. 7 . Limas CJ, Goldenberg IF, Limas C . Autoantibodies against P-adrenoceptors in human idiopathic dilated cardiomyopathy . Cire Res 1989 ;64 :97103 . 8 . Caforic, AL, Bonifaclo E . Stewart JT, et al . Novel organ-specific circulating cardiac autoantibodies in dilated cardiomyopathy . J Am Coll Cardiol 1990;15:1527-34 . 9 . Neumann DA, Burck CL, Baughman KL, Rose NR, Herskowitz A . Circulating heart-reactive antibodies in patients with myocarditis or cardiomyopathy. J Am Coll Cardiol 1990 ; 1 6:839-46. W. Ansari AA, Wang Y-C, Danner DJ, et al . Abnormal expression of histocompatibility and mitochondrial antigens by cardiac tissue from patients with myocarditis and dilated cardiomyopathy . Am J Pathol 1991 -.139 :337-54 . 11 . Caforio ALP, Grazzini M, Mann JN1, et al . Identification of a- and p-cardiac myosin heavy chain isoforms as major autoantigens in dilated cardiomyopathy. Circulation 1992 ;85 :1734-42 . 12 . Anderson IL, Carlquist JF, Hamm,)nd EH . Deficient natural killer cell activity in patients with idiopathic dilated cardiomyopathy . Lancet 1982 ; 2 :1124-7 . 13 . Fowles RE, Bieber CP, Stinson EB . Defect ;ve in vitro suppressor cell function in idiopathic congestive cardiomyopathy . Circulation 1979 ;59: 483-91 . 14. Gerli R, Rambotti P, Spinozzi F, et al . Immunologic studies of peripheral blood from patients with idiopathic dilated cardiomyopathy . Am Heart J 1986;112 :350-5 . 15 . Wong CV, Woodruff JJ . Woodruff JFR . Generation of cytoloxic T lymphocytes during coxackievirus B-3 infection : II . Characterization of effector cells and demonstration of cytotoxicity against viral-infected myofibers . J Immunol 1977 ;118 :1165-9. 16. Limas CJ, Limas C, Kubo SH, Olivari MT . Anti-beta-receptor antibodies in dilated cardiomyopathy and correlation with HLA-DR antigens . Am J Cardiol 1990,65 :483-7. 17 . Carlquist JF, Menlove RL, Muray MB, O'Connell JB, Anderson JL . HLA class 11 (DR and DQ) antigen association in idiopathic dilated cardiomyopathy : validation study and meta analysis of published HLA association studies . Circulation 1991 ;83 :515-22 . 18 . Margossian S8, White HD, Caulfield JB, Norton P, Taylor S, Slayter HS . Light chain 2 profile and activity of human ventricular myosin during dilated cardiomyopathy . Identification of a causal agent for impaired myocardial function . Circulation 1992 .85 :1720-33 . 19 . Michels VV, Driscoll DJ, Miller FA Jr . Familial aggregation of idiopathic dilated cardiomyopathy . Am J Cardiol 1985 ;55 :1232-3 . 20 . Berko BA, Swift M . X-linked dilated cardiomyopathy . New Engi J Med 1987 ;316 :1186-91 . 21 . Valantine HA, Hunt SA . Fowler MB, Billingham ME, Schroeder JS . Frequency of familial nature of dilated cardiomyopathy and usefulness of cardiac transplantation in this subset . Am J Cardiol 1989 ;63 :959-63 . 22 . Michels VV, Moll PP, Miller FA, et al . The frequency of familial dilated cardiomyopathy in a series of patients with idiopathic dilated cardiomyopathy. New En8l J Med 1992 ;326 :77-82 . 23 . Ozawa T, Tanaka M, Sugiyama S, et al . Multiple mitochondrial DNA deletions exist in cardiomyocytcs of patients with hypertrophic or dilated cardiomyopathy . Biochem Biophys Res Commun 1990 ;170 :830-6 . 24 . Tanaka M, Ino H, Ohno K, et al . Mitochondrial mutation in fatal infantile cardiomyopathy [abstract] . Lancet 1990 ;336 :1452 . 25 . Hattori K, Ogawa T, Kondo T, et al . Cardiomyopathy with mitochondrial DNA mutations . Am Heart J 1991 :122:866-9 . 26. Obayashi T, Hattori K, Sugiyama S, et al . Point mutations in mitochon-



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REMES ET AL . MITOCHONDRIAL DNA DELETIONS IN ENDOMYOCARDRJM

drial DNA in patients with hypertrophic cardiomyopathy . Am Heart J 1992 ;124:1263-9. 27 . Suomalainen A, Pactau A, Leinonen H, Majander A, Peltonen L, Somer H . Inherited idiopathic dilated cardiomyopathy with multiple deletions of mitochondrial DNA. Lancet 1992 ;W:1319-20. 28. Anderson S, Bankier AT, Barrel) BG, et al . Sequence and organization of the human mitochondrial genome . Nature 1981 ;290 :457-65 . 29 . Lentz RW, Harrison CE Jr, Dewey JD, Barnhorst DA, Danielson GK . Pluth JR . Functional evaluation of cardiac sarcoplasmic reticulum and mitochondria in human pathologic states . J Mol Cell Cardiol 1978 ;61 : 70-6. 30. Unveiferth DV, Lee SW, Wallick ET . Human myocardial adenosine triphosphatase activities in health and heart failure . Am Heart J 1988 ;115: 139-46. 31 . Holt U . Harding AE, Cooper JM, et al . Mitochondrial myopathies : clinical and biochemical features of 30 patients with major deletions of muscle mitochoWrial DNA. Ann Neurol 1989 ;26:699-708 . 32 . Hattori K, Tanaka M, Sugiyama S, et al . Age-dependent increase in the deleted mitochondrial DNA in the human heart : possible contributory factor to presbycardia . Am Heart J 1991 ;121 :1735-42 . 33 . Remes AM, Hassinen IE, MWamaa K, Peuhkurinen KJ . Mitochondrial DNA deletion diagnosed by analysis of an endomyocardial biopsy specimen from a patient with Kearns-Sayre syndrome and complete heart block . Br Heart J 1992-,68 :408-11 . 34. Linnam A, Mafzuki S . Ozawa T. Tanaka M . Mitochondria! DNA mutations as an important contributor to ageing and degenerative diseases. Lancet 1989 ;1 :642-5. 35 . Linnane AW, Baumer A, Maxwell RJ, Preston H, Zhang C, Marzuki S . Mitochondrial gene mutation : the ageing process and degenerative discases . Biochem Int 1990 ;22 :1067-76, 36. Cortopassi GA, Arnheim N. Detection of a specific mitochondria) DNA deletion in tissues of older humans . Nucleic Acids Res 1990 ;18 :6927-33 . 37 . Yen T-C, Su J-H, King K-L, Wei Y-H . Ageing-associated 5 kb deletion in human liver mitochondrial DNA . Biochem Biophys Res Common 1991 178:124-31 . 38 . Sugiyama S, Hattori K, Hayakawa M, Ozawa T . Quantitative analysis of age-associated accumulation of mitochondrial DNA with deletion in human hearts . Biochem Biophys Res Commun 1991-.180 :894-9 . 39 . Maniatis T, Fritsch EF, Sambrook J . Molecular Cloning: A Laboratory Manual. Cold Spring Harbor (NY) : Cold Spring Harbor Laboratory, 1982 :280-1,365-7. 40 . Saiki RK, Gelfand DH, Stoffel SA . et al . Primer-directed enzymatic amplification of DNA with thermostable DNA polymerase . Science 1988 ;239 :487-91 . 41 . Casanova J-L . Pannetier C . Jaulin C, Kourilsky P. Optimal conditions for directly sequencing double-stranded PCR products with Sequenase [abstract] . Nucleic Acids Res 1990 :18.Q&

JACC Vol. 23, No. 4 March 15, 1994 :935-42

42 . Hosman DN, Lemeshow S . Applied Logistic Regression . New York : Wiley, 1989. 13 . Zeviani M, Moraes CT, DiMauro S, et al . Deletions of mitochondrial DNA in Kearns-Sayre syndrome . Neurology 1988;38:1339-46. 44 . Shanske S, Mo-aes CT, Lombes A, et al . Widespread tissue distribution of mitochondria! DNA deletions in Kearns-Sayre syndrome . Neurology 1990 ;40:24-8 . 45 . Zeviani M, Genera C, Pannacci M, et al . Tissue distribution and transmission of mitochondrial DNA deletions in mitochondrial myopathies . Ann Neural 1990;28.94-7 . 46 . Corral-Debrinski M, Stephen 6, Shoffner JM, Lott MT . Kanter K, Wallace DC . Hypoxemia is associated with mitochondrial DNA damage and gene induction . Implication for cardiac disease . JAMA 1991 :266:1812-6 . 47. Brown WM . George M Jr, Wilson AC . Rapid evolution of animal mitochonihial DNA. Proc Nail Acad Sci USA 1979,76 :1%7-71 . 48. Kadenbach B, Moller-Rocker J . Mutations of milochondrial DNA and human death . Nalurwissenschaften 1990 ;77:221-5. 49. Richter C . Pro-oxidants and mitochondrial Cal'-their relationship to apoptosis and oncogenesis . FEBS Lett 199 .3 ;325 :104-7 . 50. Tepper CG, Studzinski GP . Resistance of mitochondrial DNA to degradation characterizes the apoptotic but not the necrotic mode of human leukemia cell death. J Cell Biochem 1993 ;52 :352-61 . 51 . Uppal SC . Kearns' syndrome, a new form of cardiomyopathy . Br Heart J 1973 ;35 :766-9. 52 . Roberts NK, Perloff JK . Kark PAP. Cardiac conduction in the KeamsSayre syndrome (a neuromuscular disorder associated with progressive external ophthalmoplegia anti pigmentary retinopathy) . Am J Cardiol 1979-,44 :1396-40). 53 . Kenny D, Wetherbee J . Kearns-Sayre syndrome in the elderly : mitochondrial myopathy with advanced heart block . Am Heart J 1990 .120:440-3 . 54 . Mastalgia FL, Thompson PL . Papadimitrion JM. Mitochondial myopathy with cardiomyopaby, lactic acidosis and response to prednisone and thiamine . Austr N Z I Med 1980,10:660-4. 51 Charmer KS, Channer X . Campbell M) . Russel Rees J. Cardiomyopathy in the Kearns-Sayre syndrome . Br Heart J 1988 ;59:486-90 . 56 . Schaper J, Froede R, Hein ST . et al . Impairment of myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy . Circulation 190,1,83 :504-14 . 57. Rijtig A, Colonna M, Bonnefont JP, et al . Mitochondrial DNA deletion in Pearson's marrow/pancreas syndrome . Lancet 1989 .1 :902-3. 58. Thyagarajan D, Byrne E . Noer S, et al . Significance of mitochondrial DNA deletions in myotonic dystrophy. Acta Neurol Scand 1993,87 :37-6. 59. 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 .