Journal of the American College of Cardiology
© 2002 by the American College of Cardiology Foundation
Published by Elsevier Science Inc.
Vol. 40, No. 12, 2002
ISSN 0735-1097/02/$22.00
PII S0735-1097(02)02600-1
Heart Failure
Dysfunction of Mitochondrial Respiratory Chain
Complex I in Human Failing Myocardium Is
Not Due to Disturbed Mitochondrial Gene Expression
Robert J. Scheubel, MD,*§ Mike Tostlebe, MS,§ Andreas Simm, PHD,* Susanne Rohrbach, MD,§
Roland Prondzinsky, MD,† Frank N. Gellerich, PHD,‡ Rolf-Edgar Silber, MD,* Juergen Holtz, MD§
Halle/Saale, Germany
Activity of mitochondrial respiratory chain complexes with and without mitochondrially
encoded subunits was assessed in failing human myocardium together with parameters of
mitochondrial gene expression.
BACKGROUND Mutations and deletions in mitochondrial genome (mtDNA) sporadically accumulate in the
aging myocardium. In experimental heart failure, they are discussed to be a generalized
problem resulting in disturbances of mitochondrial gene expression and mitochondrial
function.
METHODS
In left ventricular specimens from 43 explanted failing hearts and 10 donor hearts, enzyme
activities of respiratory chain complexes, messenger ribonucleic acid (mRNA) expression of
mitochondrially and nuclear encoded mitochondrial components (reverse transcriptasepolymerase chain reaction, Northern blot), undeleted wildtype mtDNA (Southern blot), and
nuclear encoded mitochondrial transcription factor A (mtTFA) (Western blot) were
quantified.
RESULTS
Citrate synthase normalized activity of mitochondrial respiratory chain complex I, which
contains seven mitochondrially encoded subunits, was decreased by 28% in terminally failing
myocardium, whereas the activity of the exclusively nuclear encoded complex II was
unchanged. However, the amount of intact mtDNA, the mRNA of all mitochondrially
encoded subunits of the entire respiratory chain, the amount of mtTFA, and the enzymatic
activity of complex III and complex IV, which also contain mitochondrially encoded subunits,
were normal compared with donor hearts, excluding generalized disturbance of mitochondrial
gene expression. Retrospective analysis of drug therapy before transplantation identified
beta-blockers as one putative protection against this disturbance.
CONCLUSIONS In terminally failing human myocardium of patients receiving drug therapy, complex I
depression is not caused by mtDNA damage and disturbed mitochondrial gene expression.
The absence of mtDNA damage should facilitate recovery of the overloaded myocardium, if
effective unloading could be achieved. (J Am Coll Cardiol 2002;40:2174 – 81) © 2002 by
the American College of Cardiology Foundation
OBJECTIVES
Hereditary or prenatally acquired mitochondriopathies,
characterized by mitochondrial deoxyribonucleic acid
(mtDNA) deletions and/or mitochondrial transfer ribonucleic acid (RNA) point mutations, are associated with
peripheral myopathies or with multisystemic syndromes,
sometimes including the myocardium with features of
dilated cardiomyopathy (DCM) (1). Affected cells show
disturbed aerobic energy production, enhanced oxidative
stress, and exaggerated susceptibility for apoptosis. In a
thorough analysis of 601 patients with DCM, 19 cases of
heteroplasmic mtDNA mutations or deletions were found
with depressed respiratory activity, suggesting that mitochondriopathies are rather rare among patients diagnosed
From the Departments of *Cardio-Thoracic Surgery, †Cardiology, ‡Neurology and
the §Institute of Pathophysiology, Martin Luther University Halle-Wittenberg,
Halle/Saale, Germany. This work was supported by grants of the German
Bundesministerium für Bildung und Forschung, BMBF (NBL 3, FKZ 1/5) and of
the DFG (SFB 598/B1).
Manuscript received May 7, 2002; revised manuscript received July 24, 2002,
accepted August 11, 2002.
for DCMs (2). In affected cases, activity of complex II,
which contains only subunits encoded by the nucleus, was
normal, whereas activities of complexes I and IV, which
contain also mitochondrially encoded subunits, were depressed, yielding a disproportional respiratory chain (2).
Secondary disturbances of mitochondrial gene expression,
not resulting from hereditary mtDNA defects, but with
similarly disproportional respiratory chains, were also found
in the glomeruli of an hereditary form of nephrotic syndrome (3) and sporadically in cells of aging organs with
minimal cellular turnover (4). While the role of such
secondary disturbances in mitochondrial gene expression in
the process of aging of postmitotic cells is still under debate,
it is remarkable that a severe, secondary disturbance of
mitochondrial gene expression has been documented in the
surviving myocardium of mice with postinfarction cardiac
failure (5). In this model, the failing myocardium exhibited
severely depressed mitochondrial gene expression, a disturbed mitochondrial respiratory chain, and substantial
losses of undeleted mitochondrial wildtype deoxyribonucleic
JACC Vol. 40, No. 12, 2002
December 18, 2002:2174–81
Abbreviations and Acronyms
ACE
⫽ angiotensin-converting enzyme
CAD
⫽ coronary artery disease
COX
⫽ cytochrome oxidase
DCM
⫽ dilated cardiomyopathy
mRNA ⫽ messenger ribonucleic acid
mtDNA ⫽ mitochondrial deoxyribonucleic acid
mtRNA ⫽ mitochondrial ribonucleic acid
mtTFA ⫽ mitochondrial transcription factor A
NADH ⫽ nicotinamide adenine dinucleotide
NO
⫽ nitric oxide
PCR
⫽ polymerase chain reaction
RNA
⫽ ribonucleic acid
rRNA
⫽ ribosomal ribonucleic acid
RT-PCR ⫽ reverse transcriptase-polymerase chain
reaction
TNF
⫽ tumor necrosis factor
acid (DNA) (5). Such secondary disturbances would not
have been detectable in human hearts by the techniques
applied in the analysis of Arbustini et al. (2). Therefore, we
performed analyses of mitochondrial gene expression in
ventricular myocardium from a nonselected series of explanted hearts from patients with terminal heart failure and
in ventricular myocardium from nonfailing donor hearts.
Using all parameters of mitochondrial gene expression
applied in the experimental mice model (5), we demonstrate
that there is no disturbance in mitochondrial gene expression in the failing explanted human myocardium from
patients who had been undergoing current drug treatment
for heart failure before explantation.
METHODS
Myocardial specimens from cardiomyopathic and donor
hearts. Samples from 10 organ donors (mean age 44.8 ⫾ 4
years), whose hearts were not suitable for transplantation
because of clinical reasons and who had no history of heart
disease, were obtained in cardioplegic arrest. In addition, 43
explanted hearts were obtained from patients (mean age 52
⫾ 1 year) undergoing transplantation due to DCM (n ⫽ 23)
or coronary artery disease (CAD) (n ⫽ 20), with a mean
ejection fraction of 24 ⫾ 1%, a mean pulmonary wedge
pressure of 20 ⫾ 1 mm Hg, and a mean cardiac index of
2.27 ⫾ 0.1 l/min/m2. Samples were immediately snap
frozen in liquid nitrogen. Twenty-five of the failing patients
had beta-blocker therapy—in 19 cases in combination with
an angiotensin-converting enzyme (ACE) inhibitor—while
the 18 patients without beta-blocker therapy received an
ACE inhibitor in 17 cases. Nine patients were treated with
a hepatic hydroxymethylglutaryl coenzyme A reductase
inhibitor (statin). No patient required positive inotropic
therapy in the form of phosphodiesterase inhibitors or
catecholamines. Samples from all 53 hearts were used for
measurement of mitochondrial respiratory chain enzyme
activity and reverse transcriptase-polymerase chain reaction
(RT-PCR). Out of 43 failing hearts, 12 were used for
Scheubel et al.
Respiratory Chain Function in Failing Heart
2175
Southern blot and 9 for Northern blot and Western blot
analysis, whereas 6 of the 10 donor hearts were used for the
three mentioned analyses.
Furthermore, out of a series of diagnostic left ventricular
biopsies performed for evaluation of causes of arrhythmias,
left ventricular biopsies without histologic signs of local
inflammation were available from three patients without
dilative cardiomyopathy. Semiquantitative RT-PCR was
performed in these biopsies.
The local ethics committee approved the study of these
human cardiac tissues, and the subjects or their families gave
informed consent.
RNA extraction and semiquantitative RT-PCR. Total
RNA was isolated from ventricular specimens, and semiquantitative RT-PCR was performed in accordance with
the protocol described previously (6). Primer sequences and
specific characteristics of semiquantitative messenger ribonucleic acid (mRNA) analyses of the analyzed genes for
ND1, ND2, ND3, ND4, ND4L, ND5, ND6, cytochrome
b, cytochrome oxidase I (COX I), COX III, 16S ribosomal
ribonucleic acid (rRNA), tumor necrosis factor (TNF)alpha, inducible nitric oxide synthase, interleukin-6, atrial
natriuretic peptide, brain natriuretic peptide, and manganese superoxide dismutase are available from the corresponding author. The polymerase chain reaction (PCR)
products were quantified per 18S rRNA.
Northern blot analysis. Total RNA from left ventricular
specimens was also used to assess mRNA levels of ND1,
ND5, cytochrome b, COX I, COX III, and 16S rRNA by
Northern blot analysis. The mRNA levels were normalized
to 18S rRNA. Ten micrograms of total RNA per lane were
separated by electrophoresis through 1.2% agarose gels after
denaturation of the RNA with glyoxal and dimethylsulfoxide. The RNA was transferred to Hybond-N⫹ nylon
membranes (Amersham Life Science Inc., Freiburg, Germany) by capillary blot with 20⫻ saline sodium citrate
buffer. The hybridization of digoxigenin-11-2⬘-deoxyuridine-5⬘-triphosphate labeled probes of PCR products
mentioned earlier was performed at 50°C according to the
protocol of the PCR DIG Probe Synthesis Kit (Roche
Diagnostics GmbH, Mannheim, Germany). Blots were
developed by enhanced chemiluminescence (Amersham
Pharmacia Biotech, Freiburg, Germany), and the intensity
of hybridizing bands was quantified by AIDA imaging
software (raytest, Berlin, Germany).
DNA isolation and Southern blot analysis of mtDNA.
Genomic DNA and mtDNA were isolated from ventricular
specimens according to the description in the Puregene
DNA Isolation Kit (Gentra Systems, Minneapolis, Minnesota). The DNA concentrations were calculated from the
absorption at 260 nm, and DNA was digested with BamH1.
Then, 3.5 ␮g of the cleaved DNA were resolved by
electrophoresis through 0.8% agarose gel. After capillary
transfer to Hybond-N⫹ nylon membrane (Amersham Life
Science Inc.), the blot was hybridized at 50°C with
digoxigenin-11-2⬘-deoxy-uridine-5⬘-triphosphate labeled
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Scheubel et al.
Respiratory Chain Function in Failing Heart
probes of PCR products for 16S rRNA and 18S rRNA
detection according to the description in the PCR DIG
Probe Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany). Blots were developed as described previously for Northern blot.
Immunoblot analysis. Proteins were isolated from ventricular specimens and electroblotted onto nitrocellulose membrane in accordance with the protocol described previously
(7).
Membranes were blocked with 5% nonfat dry milk in
Tris-buffered saline-Tween 20 (200 mM Tris [hydroxymethyl] aminomethane, 300 mM sodium chloride, 0.1%
Tween 20; pH 7.5) and incubated with the antimitochondrial transcription factor A (mtTFA) primary
antibody (dilution 1:1,000, provided by Prof. N.-G. Larsson, Department of Medical Nutrition, Karolinska Institute, Sweden). Blots were subsequently washed in Trisbuffered saline-Tween 20 and incubated with a specific
peroxidase-coupled secondary antibody (dilution 1:10,000,
anti-rabbit immunoglobulin G– horseradish peroxidase,
Amersham Pharmacia Biotech). Bound antibodies were
detected by enhanced chemiluminescence (Amersham
Pharmacia Biotech), and the intensity of hybridizing bands
was quantified by AIDA imaging software (raytest).
Determination of enzyme activity. Small pieces of frozen
tissues were homogenized (1/30 weight per volume) in a
solution containing 50 mM of Tris buffer (pH 7.5), 100
mM potassium chloride, 5 mM MgCl2, and 1 mM ethylenediaminetetraacetic acid using a glass/glass homogenizer
(Kontes Glass Co., Vineland, New Jersey, 2 ml, 0.025 mm
clearance). Enzyme activities were assayed at 30°C spectrophotometrically using a DU 640 photometer (Beckmann
Instruments, Palo Alto, California) and related to citrate
synthase activity.
The analysis of the mitochondrial respiratory chain complexes nicotinamide adenine dinucleotide (NADH):
coenzyme Q1-oxidoreductase (complex I), NADH:
cytochrome c– oxidoreductase (complex I ⫹ III), succinate:
cytochrome c– oxidoreductase (complex II ⫹ III),
ubiquinone:cytochrome c– oxidoreductase (complex III), cytochrome c– oxidase (complex IV), and citrate synthase as
mitochondrial marker enzyme was performed in accordance
with the protocol described previously (8,9).
Data analysis. Western blots, Northern blots, Southern
blots, and RT-PCR analyses were evaluated by scanning of
blots or negatives of the gel-images using a computer-based
imaging system (AIDA). The optical density units are given
as mean ⫾ SEM. The significance of comparison of mean
values was determined by the unpaired Student t test using
a significance level of p ⬍ 0.05.
RESULTS
General observations. The clinical characteristics of the
43 heart failure patients before heart explantation demonstrated that patients with CAD (n ⫽ 20) were older (55.7 ⫾
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December 18, 2002:2174–81
1.3 years) than patients with DCM (49.0 ⫾ 1.9 years, p ⬍
0.01). The patients with DCM had a more deteriorated
cardiac function (ejection fraction ⫽ 21.8 ⫾ 1.6% vs. 27.5 ⫾
2.0% in CAD, p ⬍ 0.05; pulmonary capillary wedge
pressure ⫽ 23 ⫾ 2 mm Hg vs. 17 ⫾ 2 mm Hg, p ⫽ 0.07)
and, accordingly, an increased expression of the overload
indicating factors atrial and brain natriuretic peptide (Table
1). Cardiac index and gender ratio (21 male/2 female in
DCM and 18 male/2 female in CAD) were similar in both
groups. Patients with DCM had angiographically normal
coronary arteries in all cases. The inflammatory activation of
the failing myocardium was characterized by assessment of
the mRNA expression of cytokines and inducible nitric
oxide synthase (Table 1). There was a positive correlation
between TNF-alpha mRNA and inducible nitric oxide
synthase mRNA (r ⫽ 0.29; p ⬍ 0.05; n ⫽ 53). Compared
with the biopsy samples, the inflammatory activation in
failing myocardium was substantial, both in patients with
DCM and in patients with CAD, which has been shown
previously (10).
Mitochondrial enzyme activities. The mitochondrial
electron transport chain consists of the partially mitochondrially encoded complexes I, III, and IV and the exclusively
nuclear encoded complex II. The enzymatic activity of
complex I was 28% lower in terminally failing ventricles
than in donor organs (Fig. 1A). This decrease was also
visible in the combined activity of complexes I ⫹ III (10.9
⫾ 1.3 vs. 13.7 ⫾ 1.9 in donors; p ⫽ 0.08). In contrast, there
was no decrease in the enzyme activity of the exclusively
nuclear encoded complex II, or in the partially mitochondrially encoded enzyme complexes III and IV (Fig. 1).
There was no difference in the respiratory chain enzyme
activities between myocardium from patients with DCM or
CAD (Table 1).
mtDNA copy number, mtTFA, and mitochondrial
mRNA expression. A potential reason for the decreased
complex I enzyme activity could be a disturbed mitochondrial gene expression due to oxidative stress-induced damage with subsequent deletions in the mtDNA, as documented in a murine model of postinfarction heart failure
(5). However, there was no detectable decrease in the
concentration (copy number) of undeleted wildtype
mtDNA in failing myocardium (not shown). Similarly, the
protein expression of the mtTFA was not different between
the failing ventricles and the donor hearts (1.3 ⫾ 0.2 optical
density units vs. 1.0 ⫾ 0.1 optical density units, respectively,
p ⫽ NS). Furthermore, the mitochondrial ribonucleic acid
(mtRNA) levels (obtained by RT-PCR) of all mitochondrially encoded genes of complex I (ND1, ND2, ND3,
ND4, ND4L, ND5, and ND6), of complex III (cytochrome
b), and of complex IV (COX I, COX III) as well as of 16S
rRNA were not different between failing and donor myocardium (Fig. 2A). Northern blot analysis of selected
transcripts of mitochondrially encoded subunits, e.g. ND1
(Fig. 2B), ND5, cytochrome b, COX I, or COX III
confirmed that there was no decline of any mtRNA in
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2177
Table 1. Left Ventricular mRNA Expression (Per 18S rRNA) and Respiratory Chain Complex Activities
mRNA
Pro-ANP
Pro-BNP
TNF-␣
iNOS
IL-6
ND1
ND2
ND3
ND4
ND4L
ND5
ND6
Cytochrome b
COX I
COX III
16S rRNA
MnSOD
Enzyme activity*
Complex I
Complex I ⫹ III
Complex II ⫹ III
Complex III
Complex IV
Citrate synthase
DCM
(n ⴝ 23)
CAD
(n ⴝ 20)
p Value
DCM vs. CAD
Donor
(n ⴝ 10)
p Value
Donor vs. Failing
0.85 ⫾ 0.11
0.56 ⫾ 0.07
0.21 ⫾ 0.02
0.87 ⫾ 0.09
0.14 ⫾ 0.01
1.12 ⫾ 0.07
1.34 ⫾ 0.04
0.82 ⫾ 0.08
1.45 ⫾ 0.05
0.93 ⫾ 0.04
1.29 ⫾ 0.06
1.26 ⫾ 0.07
1.49 ⫾ 0.07
1.50 ⫾ 0.07
1.19 ⫾ 0.06
1.72 ⫾ 0.09
0.65 ⫾ 0.04
0.36 ⫾ 0.07
0.30 ⫾ 0.06
0.21 ⫾ 0.02
0.86 ⫾ 0.10
0.12 ⫾ 0.02
1.05 ⫾ 0.06
1.40 ⫾ 0.07
0.95 ⫾ 0.10
1.64 ⫾ 0.10
0.91 ⫾ 0.06
1.16 ⫾ 0.05
1.40 ⫾ 0.08
1.46 ⫾ 0.06
1.39 ⫾ 0.05
1.11 ⫾ 0.04
1.61 ⫾ 0.06
0.63 ⫾ 0.03
⬍0.001
⬍0.01
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
0.15 ⫾ 0.05
0.23 ⫾ 0.08
0.15 ⫾ 0.01
0.70 ⫾ 0.07
0.09 ⫾ 0.01
1.09 ⫾ 0.09
1.41 ⫾ 0.10
0.92 ⫾ 0.09
1.46 ⫾ 0.08
0.88 ⫾ 0.08
1.20 ⫾ 0.07
1.19 ⫾ 0.10
1.38 ⫾ 0.12
1.44 ⫾ 0.09
1.02 ⫾ 0.09
1.62 ⫾ 0.11
0.64 ⫾ 0.05
⬍0.01
0.05
⬍0.05
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
11.3 ⫾ 0.56
11.3 ⫾ 0.95
20.0 ⫾ 1.19
100.6 ⫾ 6.19
41.1 ⫾ 3.03
40.5 ⫾ 1.70
11.7 ⫾ 0.66
10.5 ⫾ 0.93
18.3 ⫾ 1.57
93.4 ⫾ 5.28
39.9 ⫾ 3.62
39.6 ⫾ 2.69
NS
NS
NS
NS
NS
NS
15.6 ⫾ 1.27
13.7 ⫾ 1.44
18.4 ⫾ 0.79
98.2 ⫾ 5.17
46.4 ⫾ 3.49
44.5 ⫾ 1.51
⬍0.001
NS
NS
NS
NS
NS
*Complex activities in % of citrate synthase (Units/g/ww). Data are given as mean ⫾ SEM.
CAD ⫽ end-stage coronary artery disease; COX ⫽ cytochrome oxidase; DCM ⫽ dilated cardiomyopathy; IL-6 ⫽ interleukin-6; iNOS ⫽ inducible nitric oxide synthase;
MnSOD ⫽ manganese superoxide dismutase; mRNA ⫽ messenger ribonucleic acid; ND ⫽ ; NS ⫽ not significant; Pro-ANP ⫽ atrial natriuretic peptide; Pro-BNP ⫽ brain
natriuretic peptide; rRNA ⫽ ribosomal RNA; TNF-␣ ⫽ tumor necrosis factor alpha.
failing myocardium. Similarly, the mRNA expression of
manganese superoxide dismutase, a typical example for a
nuclear encoded mitochondrial matrix enzyme, was not
lowered in failing myocardium (Fig. 2A). In the left
ventricular biopsies without histologic signs of inflammation, relative interleukin-6 mRNA expression (0.03 ⫾ 0.01
U) was lower (p ⫽ 0.07) than in donor hearts (0.09 ⫾ 0.01
Figure 1. Enzymatic activity of electron transport chain complexes I (A), II
⫹ III (B), III (C), and IV (D) normalized to citrate synthase activity (CS)
in human left ventricular myocardium of terminally failing hearts (n ⫽ 43)
and donor hearts (n ⫽ 10). Data are given as mean ⫾ SEM. ***p ⬍ 0.001.
U, Table 1). However, mRNA expression of ND1, ND5, or
cytochrome b and of the nuclear encoded gene manganese
superoxide dismutase was not lowered (data not shown). In
failing myocardium, there was no difference in any mtRNA
concentration between DCM and CAD (Table 1).
Effects of drug treatment on mitochondrial enzyme
activity and gene expression. The retrospective analysis of
terminally failing explanted hearts from patients undergoing
beta-blocker therapy (n ⫽ 25) before explantation and those
without this therapy (n ⫽ 18) showed that both groups had
a similar degree of cardiac overload, indicated by the
fourfold elevation of atrial natriuretic peptide mRNA expression in both groups (0.66 ⫾ 0.18 in beta-blocker–free
patients vs. 0.61 ⫾ 0.16 in beta-blocker–treated patients
compared with donors [0.15 ⫾ 0.07; p ⬍ 0.05]). Furthermore, both subgroups had no differences in age, ejection
fraction, pulmonary wedge pressure, and cardiac index.
However, the beta-blocker–treated patients had a 20%
higher complex III enzyme activity compared with the
patients without this treatment, whereas this difference in
the other complex activities did not reach the level of
significance (Fig. 3).
Retrospective subgrouping of the terminally failing hearts
into those from patients undergoing ACE inhibitor therapy
(n ⫽ 37) before explantation and those without this therapy
(n ⫽ 6) demonstrated a lower TNF-alpha mRNA expression in patients undergoing ACE inhibition (0.20 ⫾ 0.02
2178
Scheubel et al.
Respiratory Chain Function in Failing Heart
JACC Vol. 40, No. 12, 2002
December 18, 2002:2174–81
Figure 3. Comparison of enzymatic activity of electron transport chain
complexes I (A), II ⫹ III (B), III (C), and IV (D) normalized to citrate
synthase activity (CS) in human left ventricular myocardium from terminal
heart failure patients receiving beta-blocker therapy (␤-bl. pos., n ⫽ 25)
before explantation and patients without this therapy (␤-bl. neg., n ⫽ 18).
Data are given as mean ⫾ SEM. *p ⬍ 0.05.
Figure 2. (A) Reverse transcriptase-polymerase chain reaction analysis of
messenger ribonucleic acid (mRNA) expression of mitochondrially encoded subunits of respiratory chain complexes (C-I, C-III, C-IV) in
human failing left ventricular myocardium given as percent of donor values.
Data were obtained by densitometric quantification of the negatives of
gel-images and are given as mean ⫾ SEM. (B) Representative Northern
blot analysis of mitochondrially encoded ND1 mRNA and nuclear encoded
18S ribosomal ribonucleic acid (rRNA) in left ventricular myocardium of
donors and terminally failing patients.
vs. 0.27 ⫾ 0.02 in patients without this treatment, p ⬍
0.05), but no hints of any difference in mitochondrial
complex activities or gene expression.
DISCUSSION
This study of left ventricular myocardium from explanted
hearts demonstrates that the activity of mitochondrial complex I, containing seven mitochondrially encoded subunits,
is similarly lowered in patients suffering from DCM or
ischemic heart disease. On the other hand, however, the
activity of complex II, containing exclusively subunits encoded by the nucleus, is unchanged in both groups of
patients. At first glance, this result appears compatible with
dysfunction of mtDNA expression, as has been shown
previously in a murine postinfarction model of heart failure
(5).
In that experimental study, several observations suggested
disturbed expression of the mitochondrial genome: in the
nonischemic part of failing ventricles, concentrations of
transcripts of mitochondrially encoded subunits were signif-
icantly lower, the activities of complexes III and IV (which
also contain mitochondrially encoded subunits) were even
more depressed than the activity of complex I, the amount
of the non-deleted mitochondrial wildtype DNA was depressed to 44% of control myocardium, and the ventricular
protein content of mtTFA was also depressed to 65% (5).
An enhanced radical formation was demonstrated in the
mitochondria of failing ventricles, and the loss of undeleted
wildtype mtDNA was considered as a consequence of DNA
damage by oxidative stress, resulting in lowered mitochondrial transcript availability and lowered mtTFA content,
which is degraded more rapidly in cells with a deficit of
wildtype mtDNA (11).
None of these alterations, except for the complex I
depression, could be confirmed in our study in failing
human myocardium: The content of undeleted wildtype
mtDNA (measured by Southern blotting, not shown) was
normal, as was mtTFA protein and the amount of all
mitochondrially encoded transcripts (Fig. 2A). A general
impairment in the translation of mitochondrial transcripts
in human hearts is also unlikely, because complexes III and
IV, which contain mitochondrially encoded subunits, were
normal (Fig. 1).
A critical aspect in studies on failing human myocardium
from explanted hearts is the nonfailing donor myocardium
used as control. Subsequent to brain death before organ
removal and during cardioplegia, donor hearts experience
severe inflammatory activation, indicated by enhanced expression of interleukin-6 and other cytokines (12,13). Such
activation might conceivably interfere with mitochondrial
gene expression and function. Compared with the biopsies
without histologic signs of inflammation, our donor hearts
had elevated interleukin-6 expression, compatible with
JACC Vol. 40, No. 12, 2002
December 18, 2002:2174–81
some inflammatory activation. However, mitochondrial
gene expression in these biopsies was not higher than in the
donor hearts, arguing against inflammation-associated depression of mitochondrial gene expression in our donor
hearts.
Several factors might be considered as explanations for
these striking discrepancies between human and murine
heart failure. A basic difference between heart failure in
patients and the murine model is the history of the disease
and the degree of decompensation, which is probably
maximal in the rapidly progressing experimental model with
extremely large infarcts (5). In our study, the patients with
DCM had significantly higher atrial natriuretic peptide
expression and exhibited more hemodynamic decompensation compared with patients with ischemic heart disease
(Table 1 and the “General observations” section under the
“Results” heading). However, there was no difference between these two patient groups in mitochondrial transcript
concentrations and in the activities of all mitochondrial
respiratory chain complexes (Table 1). Whether drug treatment of heart failure might have been protective against
dysfunction of mitochondrial gene expression in our patients remains open. Retrospective subgrouping of patients
according to treatment with ACE inhibition/angiotensin II
type 1 receptor blocker or without such treatment revealed
higher TNF-alpha mRNA expression in the latter group
but no differences in parameters of mitochondrial gene
expression. Retrospective subgrouping of patients according
to treatment with beta-blockers (Fig. 3) revealed some
protection of mitochondrial respiratory function, significant
only for complex III activity in beta-blocker–treated patients
(Fig. 3C). This might be considered as a vague hint of some
protective effect of beta-blocker treatment on mitochondrial
function in failing myocardium. A similar impression of
beta-blocker–mediated mitochondrial protection was previously obtained in our retrospective analysis of apoptotic
activation in human failing myocardium (7).
A putative explanation for the depression in complex I
might be derived from observations in macrophage cell lines
(14,15). In these cells, nitric oxide (NO) induces a competitive, rapidly reversible inhibition of complex IV, resulting
in enhanced radical formation by the respiratory chain
upstream this inhibition. In situations with lowered antioxidant defense, this combination of NO and enhanced
mitochondrial superoxide anion formation results in a
slowly developing, irreversible depression of complex I
activity, probably via peroxynitrite formation and/or
S-nitrosylation of not yet identified targets within complex
I, whereas other complexes are not affected (14,15). Although this sequence has not yet been analyzed in failing
myocardium, all conditions for this pattern of reactions are
given: enhanced NO formation in failing myocardium has
been documented (16) and can be assumed in the hearts in
this study with enhanced TNF-alpha expression correlating
to inducible nitric oxide synthase expression; NO is a potent
inhibitor of complex IV in myocardium (17); and enhanced
Scheubel et al.
Respiratory Chain Function in Failing Heart
2179
oxidative stress in failing myocardium due to mitochondrial
and extramitochondrial mechanisms has been documented
(18 –20).
The complex I deficiency in human failing myocardium
should have functional consequences for respiratory adenosine triphosphate production and for mitochondrial radical
formation. Inhibition of mitochondrial complex I to various
degrees and by different interventions allowed titration of
the effect of complex I deficiency for maximal respiratory
capacity (21). From these data it can be derived that the 28%
depression in complex I activity of the failing human heart
(Fig. 1) results in a 15% decline in maximal respiratory
capacity (21). This respiratory capacity is further attenuated
in the failing human heart by the mitochondrial loss of
cytochrome c (7), and probably by some reversible inhibition of complex IV by enhanced NO formation (17) (the
latter inhibition is not detectable during the applied analysis
of the complex IV activity in vitro because of NOS
inactivation due to lack of L-arginine and calcium, see
“Methods” section). Thus, several mechanisms for a primary
attenuation in mitochondrial adenosine triphosphate synthesis are now identified, which explain the reduced respiratory capacity in skinned fibers from failing human myocardium under state-3 respiration supported by substrates
for complex I (22). This reduction in mitochondrial respiratory capacity of failing myocardium is considered as causal
for the reported lowered myocardial concentrations of phosphocreatine and adenosine triphosphate (23), for enhanced
glycolytic activity (24), and it may be the reason for the
tendency toward enhanced endothelin-1 formation (25,26).
This enhanced endothelin-1 expression results from
hypoxia-inducible factor-1alpha induction due to impaired
energy metabolism and can be mimicked by complex I
inhibition (25,27). Heart failure-associated mitochondrial
alterations downstream to the respiratory chain complexes,
such as reduction in mitochondrial creatine kinase (28) and
creatine transporter (29), might probably be compensated
by an enhanced adenylate kinase– catalyzed phosphotransfer
(30,31).
Complex I deficiency due to inhibition by rotenone or
due to attenuated mitochondrial gene expression in mitochondriopathies results in enhanced mitochondrial production of superoxide anion (32,33). Similarly, in experimental
heart failure with complex I activity depressed to the same
extent as in our human failing myocardium, complex I is the
source of enhanced mitochondrial radical formation (20),
although the mechanisms of complex I deficiency appear
different.
Apart from cases of mitochondriopathies, comparisons in
respiratory chain activities between failing and non-failing
myocardium have seldom been performed. In explanted left
ventricular myocardium from patients with DCM, a dramatic depression of NADH:cytochrome c– oxidoreductase
(complex I ⫹ III activity) by 61% compared with donor
myocardium has been reported, whereas NADH:CoQ1–
oxidoreductase (complex I) was normal (34). This is difficult
2180
Scheubel et al.
Respiratory Chain Function in Failing Heart
to understand even in the presence of attenuated complex
III activity, because the NADH cytochrome c– oxidoreductase activity almost exclusively depends on complex I activity
(35). In an experimental model of tachypacing-induced
heart failure without any drug treatment, a depression in the
activities of all complexes containing mitochondrially encoded subunits, including the mitochondrial adenosine
triphosphatase, was described (36), indicating disturbed
mitochondrial gene expression similar to the data in the
murine failure model mentioned previously (5). Thus, these
heart failure models in two different species both without
drug treatment (5,36) exhibit a disproportional respiratory
chain as it results from disturbed mitochondrial gene expression (5). On the other hand, terminally failing explanted
human myocardium of patients undergoing established drug
treatment for heart failure suggests a protective role of drug
treatment against disturbances of mitochondrial gene expression.
Conclusions. Although failing human myocardium exhibits a significant deficit in the activity of complex I, this
deficit is not due to generalized damage of mtDNA and
disturbed mitochondrial gene expression. Other complexes
of the respiratory chain have normal activity regardless of
whether they contain mitochondrially encoded subunits.
The protective role of drug treatment, specifically by betablockers, against global damage of mtDNA remains to be
proven. The absence of global mtDNA damage should
facilitate recovery of the overloaded myocardium, provided
that primary causes of heart failure could be adequately
corrected.
Acknowledgments
We appreciate the cooperation of the patients and the staff
of the Clinic for Cardio-Thoracic Surgery in Halle/Saale,
Germany. We thank S. Kahrstedt, E. Land, and R. Busath
for their technical assistance. We also thank N.-G. Larsson,
Department of Medical Nutrition, Karolinska Institutet,
Huddinge Hospital, Sweden, for providing the human
mtTFA antibody.
Reprint requests and correspondence: Dr. Robert J. Scheubel,
Klinik für Herz- und Thoraxchirurgie, Martin-LutherUniversitaet Halle-Wittenberg, Ernst-Grube-Str. 40, D-06097
Halle (Saale), Germany. E-mail: [email protected].
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