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Cardiovascular Research 52 (2001) 103–110
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Abnormal cardiac and skeletal muscle mitochondrial function in
pacing-induced cardiac failure
´
´ a , *, Michael J. Goldenthal a , Gordon W. Moe b
Jose´ Marın-Garcıa
a
The Molecular Cardiology and Neuromuscular Institute, 75 Raritan Ave., Highland Park, NJ 08904, USA
b
Terrance Donnelly Heart Center, St. Michael’ s Hospital, University of Toronto, Toronto, Ont., Canada
Received 16 February 2001; accepted 23 May 2001
Abstract
Background: Previous studies have shown that marked changes in myocardial mitochondrial structure and function occur in human
cardiac failure. To further understand the cellular events and to clarify their role in the pathology of cardiac failure, we have examined
mitochondrial enzymatic function and peptide content, and mitochondrial DNA (mtDNA) integrity in a canine model of pacing-induced
cardiac failure. Methods: Myocardium and skeletal muscle tissues were evaluated for levels of respiratory complex I–V and citrate
synthase activities, large-scale mtDNA deletions as well as peptide content of specific mitochondrial enzyme subunits. Levels of
circulating and cardiac tumor necrosis factor-alpha (TNF-a), and of total aldehyde content in left ventricle were also assessed. Results:
Specific activity levels of complex III and V were significantly lower in both myocardial and skeletal muscle tissues of paced animals
compared to controls. In contrast, activity levels of complex I, II, IV and citrate synthase were unchanged, as was the peptide content of
specific mitochondrial enzyme subunits. Large-scale mtDNA deletions were found to be more likely present in myocardial tissue of paced
as compared to control animals, albeit at a relatively low proportion of mtDNA molecules (,0.01% of wild-type). In addition, the
reduction in complex III and V activities was correlated with elevated plasma and cardiac TNF-a levels. Significant increases in left
ventricle aldehyde levels were also found. Conclusions: Our data show reductions in specific mitochondrial respiratory enzyme activities
in pacing-induced heart failure which is not likely due to overall decreases in mitochondrial number, or necrosis. Our findings suggest a
role for mitochondrial dysfunction in the pathogenesis of cardiac failure and may indicate a commonality in the signaling for
pacing-induced mitochondrial dysfunction in myocardial and skeletal muscle. Increased levels of TNF-a and oxidative stress appear to
play a contributory role.  2001 Elsevier Science B.V. All rights reserved.
Keywords: Energy metabolism; Heart failure; Mitochondria
1. Introduction
Heart failure is a chronic disorder characterized by a
relentless progressive clinical course often resulting in
repeated hospital admissions which imposes a burden on
the health care delivery system [1]. Accordingly, further
understanding of the mechanisms mediating the progression of heart failure is of major importance. Although
hemodynamic stress frequently encountered in heart failure
may play a role in the disease progression [2], it is
currently believed that many other mechanisms such as the
*Corresponding author. Tel.: 11-732-220-1719; fax: 11-732-2202992.
´
´
E-mail address: [email protected] (J. Marın-Garcıa).
activation of neurohormones and the pro-inflammatory
cytokines may be more important in mediating the progression of heart failure [3,4]. Recent studies with isolated
cells including cardiomyocytes have implicated the effects
of pro-inflammatory cytokines tumor necrosis factor-alpha
(TNF-a) in the generation of reactive oxygen species
(ROS) in mitochondria, altered mitochondrial membrane
permeability and in mitochondrial enzymatic dysfunction
as both early events and critical to the physiological
mechanism of TNF-a action [5–7].
A potentially important and yet poorly studied mechanism for the progression of chronic heart failure (CHF) is
perturbed myocardial energetics which have been reported
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PII: S0008-6363( 01 )00368-6
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´ et al. / Cardiovascular Research 52 (2001) 103 – 110
J. Marın-Garcıa
in models of heart failure or cardiac hypertrophy [8].
Indeed, discrete mitochondrial oxidative phosphorylation
(OXPHOS) defects have been documented in human
cardiomyopathies. Both dilated and hypertrophic cardiomyopathies are frequently accompanied by changes in
OXPHOS / respiratory enzyme activities in cardiac tissues
[9–11]. Also, large-scale deletions in heart mitochondrial
DNA (mtDNA) have been reported in patients with
cardiomyopathy [12–14]. Furthermore, changes in bioenergetics and in both mitochondrial structure and respiratory
enzyme function have been described in skeletal muscle in
CHF [15–17]. However, most of these studies have
involved patients with heart failure with diverse etiologies,
which would invariably confound the interpretation of the
energetic data. Accordingly, to study changes in mitochondrial function in heart failure per se without the
confounding effects of such as myocardial ischemia, would
require a relatively pure animal model of heart failure.
Canine pacing-induced cardiomyopathy is a model of heart
failure in which rapid ventricular pacing leads to cardiac
myopathy consisting of an increase in chamber dimension,
mural thinning, elevation in ventricular wall stress, and
congestive heart failure, mimicking dilated cardiomyopathy in humans [18–21].
In our previous studies, myocardial tissues from paced
dogs were found to harbor markedly reduced activities of
key ATP-utilizing and ATP generating enzymes including
mitochondrial ATP synthase [19]. However, the
pathophysiologic importance of mitochondrial enzyme
activity reductions in mediating the progression of heart
failure remains undetermined. Bioenergetic and mitochondrial enzyme dysfunction could play an integral part in the
primary mechanism of cardiac dysfunction or may represent a common downstream event in the pathways leading
to the heart failure phenotype. Data gauging the extent to
which mitochondrial abnormalities are either a primary
defect or are secondary to other myocardial changes (e.g.
cardiac hypertrophy and remodeling) that contribute to the
pathophysiology of cardiac cell function are needed. To
assess the extent and tissue-specificity of mitochondrial
dysfunction in heart failure, we therefore conducted a
comprehensive analysis of mitochondrial respiratory enzymatic activities and of mtDNA integrity in both the
myocardial and skeletal muscle of dogs with severe
pacing-induced heart failure as compared to tissues from
normal animals. To evaluate the potential effects of
cytokine activation and oxidative stress, levels of both
circulating and cardiac-localized TNF-a and of overall
aldehyde content were evaluated.
2. Methods
The study population consisted of two groups of dogs. A
total of ten normal dogs served as controls. A second
group of 11 dogs underwent continuous rapid right ven-
tricular pacing for 3 weeks and was designated as the
group with heart failure. Approval was obtained from the
institutional animal research committee before study commencement, in accordance with the guidelines on the Care
and Use of Experimental Animals issued by the Canadian
Council on Animal Care, Ottawa, Canada.
2.1. In vivo studies
The method of induction of heart failure and the clinical
and in vivo hemodynamic assessment have been described
in detail in previous reports [18–20]. In brief, under
general anesthesia, a bipolar lead was placed in the right
ventricle and a programmable pulse generator (SX-5985,
Medtronic, Missisauga, Ont., Canada) was placed in a
cervical pocket. A chronic indwelling cannula was placed
in the carotid artery for serial blood sampling. Dogs were
recovered from anesthesia for 1 week before the first in
vivo study. After baseline radiographic and hemodynamic
assessment to ensure healthy baseline parameters, the
pacemaker was programmed to deliver impulses at 250
beats / min. The animals were examined weekly for clinical
and radiographic signs of heart failure. Arterial blood
sampling was performed weekly to monitor changes in
plasma cytokine levels. All in vivo measurements were
obtained with the conditioned animal in a conscious state.
In paced dogs, data were acquired after sinus rhythm was
resumed temporarily for 15 min to eliminate the artifacts
produced by pacing. Hemodynamic measurements were
obtained using a micromanometer-tipped catheter introduced via the femoral artery and a thermodilution catheter
positioned in the pulmonary artery introduced via the
femoral vein. Measurements were obtained at baseline and
after 3 weeks of pacing. For the terminal hemodynamic
study, the data were acquired after the pacer was programmed off for 20 min. After the study, the dogs were
euthanized and tissue from the lateral wall of the left
ventricle and skeletal muscle from the vastus lateralis,
|500 mg of each, were obtained and stored at 2808C until
analysis.
2.2. Enzymology
Tissues were analyzed for mitochondrial enzyme activities using tissue homogenates prepared in MTE buffer
(0.25 M mannitol, 20 mM Tris–S0 4 and 1 mM EDTA, pH
7.4) with a glass homogenizer. Complex I activity measured by the oxidation of NADH by ubiquinone-1 at 340
nm, complex II activity measured by the oxidation of
succinate by ubiquinone-2 at 600 nm, complex III assayed
by reduction of cytochrome c by ubiquinol-2 at 550 nm,
complex IV assayed by oxidation of dithionite-reduced
cytochrome c at 550 nm, complex V assayed by NADH
oxidation using the coupled enzyme assay with pyruvate
kinase and lactate dehydrogenase at 340 nm, and citrate
synthase assayed at 412 nm were all performed spectro-
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J. Marın-Garcıa
photometrically at room temperature [9,10]. In all cases
duplicate assays were performed. Protein determination
was performed according to Bradford [22].
2.3. Immunoblot analysis of specific mitochondrial
proteins
105
mean value of three separate measurements performed in
duplicate. The buffer used for solubilizing the membrane
bound protein was used as negative control to confirm that
homogenizing buffer did not interfere with the ELISA
result.
2.6. Oxidative stress
Samples of crude homogenate protein (20–100 mg)
were denatured in buffer containing 2% sodium dodecyl
sulfate (SDS), 62.5 mM Tris–HCl, pH 6.8, and 10%
glycerol, and incubated before electrophoresis for 5 min at
1008C with b-mercaptoethanol. SDS polyacrylamide gel
electrophoresis was carried out at 100 V using 4–20%
acrylamide (Tris–glycine) gradient gels. The proteins were
then transferred electrophoretically to Immobilon P membranes (Millipore) in Tris–glycine buffer containing 10%
methanol for 1 h at 100 V. After blocking the membrane
with phosphate buffered saline with 0.05% Tween-20 and
3% (w / v) instant Carnation dry milk for 60 min, the blots
were incubated for 1 h at room temperature with antibody
against either cytochrome c (PharmGen), heat shock
protein (HSP-60) (StressGen) or subunits a and g of
human ATP synthase kindly provided by M.F. Marusich
(University of Oregon, Eugene). A detection system
employing alkaline phosphatase conjugated to an appropriate second antibody, and colorimetric substrate (NBTBCIP) was used (Roche). Quantitation of the signals was
performed on scanned blots using Image 1.33 software
(NIH).
2.4. PCR amplification
DNA was extracted from cardiac homogenates using
QIAamp Tissue kit (Qiagen) and amplified by PCR using
primers of specific canine mtDNA sequences [23] designed
using Right Primer software (BioDisk). PCR amplification
was performed in 100 ml total volume with 200 mM of
each dNTP, 0.4 mM of each primer, 1 U of Taq polymerase
(Promega), 2.0 mM MgCl 2 , 10 mM Tris–HCl pH 8, with
50 mM KCl and 100 ng of DNA template. After heating at
948C for 3 min to ensure DNA denaturation, each cycle of
PCR consisted of denaturation at 948C for 60 s, annealing
at 548C for 60 s and extension at 728C for 60 s. After 35
cycles of the PCR reaction and an additional extension step
at 728C for 8 min, 15 ml of the reaction mixture was
electrophoresed in a 2.5% agarose gel and stained with
ethidium bromide.
2.5. Tumor necrosis factor-a
Quantitative analysis of serum and tissue tumor necrosis
factor-a (TNF-a) was performed with ‘sandwich’ enzyme
linked immunoadsorbant assay (ELISA) using a commercially available Mouse TNF-a DuoSet kit (Genezyme
Diagnostics, Cambridge, MA). The antibodies cross-react
with human, dog, and rat TNF-a. Results represented the
Total aldehydes (expressed as pmol / 100 mg tissue) were
measured in left ventricular tissues using gas chromatography–mass spectrometry [24].
2.7. Statistics
Data concerning enzyme activity, hemodynamic measurements, plasma and cardiac TNF-a levels, aldehyde and
peptide content, were subjected to an unpaired t-test.
Differences were considered significant when the calculated probability value was less than 0.05.
3. Results
Development of heart failure in paced dogs was confirmed by clinical, radiographic and hemodynamic assessments. In the paced group, all dogs developed at least two
of the three signs of apathy, anorexia and ascites, and all
displayed severe pulmonary congestion on the chest
radiographs. The hemodynamic data of control normal
dogs and paced dogs are summarized in Table 1. In paced
dogs, heart rate, mean pulmonary arterial, pulmonary
capillary wedge, left ventricular end diastolic and right
atrial pressures were all markedly elevated compared to
normal controls. On the other hand, cardiac index and left
ventricular dP/ dt were significantly depressed. Mean
arterial pressure was unaffected in paced dogs. Data for
plasma TNF-a level are shown in Fig. 1. Compared to
control dogs, paced dogs had markedly elevated serum
TNF-a levels suggesting an activation of the pro-inflammatory cytokines. Levels of cardiac TNF-a were also
significantly elevated in the paced animals (171.7612.3
pg / mg tissue) compared to the controls (68.4612.3 pg / mg
tissue).
Left ventricular samples from each of 21 dog hearts (11
paced, ten non-paced controls) and skeletal muscle biopsies were analyzed for respiratory enzymatic activity levels
(complex I–V) and for levels of citrate synthase. As shown
in Table 2, specific activity levels of myocardial complex
III and V were significantly lower in paced animals
(greater than 70% reduced compared to mean values from
non-paced animals, P,0.05). Furthermore, complex III
and V activities correlated inversely with serum TNF-a
levels (r520.6631 and 20.5498, respectively, both P,
0.05)., and with cardiac TNF-a (r520.7500 and 20.7838,
respectively, both P,0.05). Levels of other mitochondrial
respiratory complex activities including complex I, II and
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106
Table 1
Hemodynamic data
LV end diastolic pressure (mmHg)
Mean pulmonary artery pressure (mmHg)
Right atrial pressure (mmHg)
Heart rate (beats / min)
Cardiac index (ml / kg)
Mean arterial pressure (mmHg)
Pulmonary capillary wedge pressure (mmHg)
Left ventricular dP/ dT (mmHg / s)
Control dogs
Paced dogs
10.960.67
16.361.02
7.460.53
75.069.07
151.7611.55
107.663.74
9.060.6
2069.56121.80
37.662.15*
39.562.4*
12.760.88*
137.466.6*
94.2268.22*
10762.18
29.162.01*
1158.5636.77*
Values shown are mean6S.E.M.
* P,0.05 versus control.
IV as well as citrate synthase activity, an enzyme often
used as a gauge of overall mitochondrial content, were not
significantly diminished in the paced compared to untreated animals. If activities were normalized with respect
to citrate synthase activity, significant reduction was noted
in the activity ratios for complex III / citrate synthase and
complex V/ citrate synthase (greater than 65% reduced
compared to control activity ratios), while activity ratios
for complex I, II and IV were not affected. Similar findings
were obtained in skeletal muscle activity levels and
normalized activity ratios for complex III and V (Table 3).
In humans, mtDNA damage can be evaluated by gauging the presence and abundance of specific mtDNA
deletions which frequently increase in overall abundance in
cardiac and skeletal muscle tissues in several cardiac and
neurological diseases as well as during the course of aging
[25,26]. Using primers designed from the known canine
mtDNA sequence [23] roughly analogous to sequences
used to evaluate the common 7.4-kb deletion in humans
[13], specific large-scale mtDNA deletions were found to
be present in |50% of paced dog cardiac tissues examined.
These deletions were not observed in the control myocardial tissues as depicted in Fig. 2. The mtDNA deletions in
the paced dog myocardium (estimated to be |7.55 kb in
size) are somewhat larger (100–200 bp) than those reported in human mtDNA but delete the same relative
regions of the mitochondrial genome as found in the
common 7.4-kb deletion in humans. They are present at
extremely low abundance (less than 0.01%) compared to
wild-type genomes (results not shown). These deletions
were not present in skeletal muscle from either paced or
non-paced dogs.
Levels of peptide content for specific mitochondrial
proteins including cytochrome c, two subunits of respiratory complex V (ATPase g and a subunits) and HSP-60 were
Table 2
Mitochondrial enzyme activities and activity ratios in myocardial tissues
Table 3
Mitochondrial enzyme activities and activity ratios in skeletal muscle
Fig. 1. TNF-a serum levels in paced-induced CHF and control animals.
The data are expressed as mean value6S.E.M. error bars.
Control dogs
a
Complex I
Complex I / CS
Complex II a
Complex II / CS
Complex III a
Complex III / CS
Complex IV a
Complex IV/ CS
Complex V a
Complex V/ CS
Citrate synthase a
25.463.2
0.04760.003
24.763.49
0.0460.01
11.262.06
0.02260.004
207.20615.19
0.3960.03
192.60624.55
0.3460.03
555.90649.65
Paced dogs
19.863.3
0.04760.006
22.763.18
0.0360.01
2.0960.73*
0.00460.002*
187.45611.98
0.3360.02
56.8268.05*
0.1060.01*
583.55640.95
Values shown are mean6S.E.M. with n510 for each group. CS, citrate
synthase.
a
Results shown correspond to the calculated mean values of specific
activity6S.E.M. All units are nmol substrate used / min per mg protein.
* P,0.05 versus control.
a
Complex I
Complex I / CS
Complex II a
Complex II / CS
Complex III a
Complex III / CS
Complex IV a
Complex IV/ CS
Complex V a
Complex V/ CS
Citrate synthase a
Control dogs
Paced dogs
16.661.1
0.04760.003
16.463.37
0.0660.01
10.5061.51
0.0460.01
142.69613.19
0.5060.05
151.20614.41
0.5160.05
314.90638.04
16.161.8
0.04760.006
16.762.96
0.0760.02
0.9060.46*
0.00460.002*
138.10614.43
0.5160.06
83.8065.39*
0.3160.03*
286.51627.03
Values shown are mean6S.E.M. with n510 for each group. CS, citrate
synthase.
a
Results shown correspond to the calculated mean values of specific
activity6S.E.M. All units are nmol substrate used / min per mg protein.
* P,0.05 versus control.
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J. Marın-Garcıa
107
Fig. 2. Large-scale deletions in mtDNA in CHF. Cardiac mtDNA was amplified using a set of dog-specific mtDNA primers analogous to human sequences
previously used to assess the presence of the common 7.4-kb mtDNA deletion [10]. While the expected amplification product with this primer set is greater
than 7.8 kb (and would not likely be amplified nor visualized under these conditions), smaller amplified fragments (sized |475 and 280 bp) indicative of
large-scale mtDNA deletions are shown on this representative agarose gel (2.5%) with the paced (P) and not with the control (C) DNA templates. Lanes
(M) containing size markers of 271, 449, 683 and 885 bp are also shown.
not significantly different in cardiac tissues of paced as
compared to control animals (Fig. 3).
As an indication of increased oxidative stress in CHF,
levels of cardiac tissue total aldehydes were markedly
increased in paced (2228461690 pmol / 100 mg tissue)
compared to control values (83986477 pmol / 100 mg
tissue). Increases were also observed in several species of
unsaturated aldehydes including 4-OH-alkenals and malondialdehyde.
4. Discussion
Abnormalities in the number, structure and function of
myocardial mitochondria have been described in a considerable number of patients with cardiomyopathy, although
such ultrastructural changes are often less specific and
diagnostic than demonstrated enzymatic activity changes
[17]. Myocardium with an increased number of smaller
mitochondria has been reported in animals with severe
heart failure including dogs with CHF produced by
sequential intracoronary microembolizations [27]. Evidence has also been presented describing myocyte loss,
and altered myocyte geometry indicating extensive
myocyte re-modelling and triggering of apoptotic pathway(s) in the canine model of pacing-induced heart failure
used in our studies [28,29]. However, although there have
been reports of mitochondrial dysfunction in cases of
human cardiomyopathy, there is at present little information available concerning the comprehensive assessment
of mitochondrial respiratory enzymatic function in both
myocardial and skeletal muscle tissues in an animal model
of heart failure. Our principal findings in this study
indicate that marked reductions in respiratory complex III
and V activities are present in both myocardial and skeletal
muscle tissues after pacing-induced cardiac failure. These
changes do not appear to be accompanied by changes in
peptide content of specific mitochondrial proteins.
Our previous studies using this model have reported
decreases in myocardial tissues of paced animals of the
major mitochondrial ATP-generating enzyme (complex V
or mitochondrial ATP synthase) as well as downregulation
of several ATP utilizing enzyme activities [19,20] associated with a possibly compensatory increase in fatty acid
oxidation and Krebs cycle activities. The reduction in the
levels of myocardial mitochondrial ATP synthase activity
has been further identified as both an early and a persistent
event during the development of heart failure [20]. Those
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J. Marın-Garcıa
Fig. 3. Peptide content of specific mitochondrial enzyme subunits in paced and control tissues. A representative Western blot probed with antibodies
directed against cytochrome c and ATP synthase subunit a (as described in Methods) is shown. The M (marker) lane contains pre-stained molecular weight
markers for SDS–PAGE (InVitrogen).
studies however were limited since other mitochondrial
enzymes involved in oxidative phosphorylation and evidence of general mitochondrial damage were not assessed.
In this study, the data confirm the marked reduction of
myocardial complex V as well as identifying a reduction in
complex III activities in the paced animals. Our results also
show no significant change, or evidence of compensatory
increase, in the activity levels of citrate synthase, the
mitochondrial-matrix located Krebs cycle enzyme. Importantly, the reduced levels of complex III and V activities
do not appear to be due to generalized mitochondrial
damage, necrosis or overall decreased levels of mitochondria as gauged by unchanged levels of respiratory
complex I, II, IV and citrate synthase activities relative to
controls, levels of mitochondrial located peptides including
cytochrome c, HSP-60 subunits a and g of complex V and
are further corroborated by data showing unchanged levels
of mtDNA in paced dog myocardium compared to control
(data not shown).
The reason for the pronounced selective reduction in
these specific enzyme activities in heart failure at present
remains undetermined. Peptide levels for two of the
nuclear-encoded subunits of ATP synthase (complex V)
were unchanged. However, reduced levels of other complex V subunits could potentially account for the reduction
in complex V activity level; other subunit peptide levels
have not been examined and should be assessed when
specific antibody probes to canine peptides become available. Moreover, future studies aimed at determining both
the specific levels of mitochondrial peptides in complex V
(e.g. ATPase 6 and ATPase 8) and complex III (e.g. cyt b)
as well as the import and assembly of the nuclear-encoded
subunits into functioning mitochondrial respiratory complexes, could prove informative in understanding the
pathophysiology of cardiac failure. In this regard, recent
studies of complex IV (cytochrome c oxidase) deficiency in
Leigh disease have demonstrated defective assembly of
complex IV subunits in several tissues [30]. Our results
also suggest that a common element in either the synthesis,
import, and / or assembly, or regulation of both complex V
and III (but not complex I or IV) subunits may be
defective, in both cardiac and skeletal muscle in canine
pacing-induced heart failure. Defective regulation of mitochondrial transcription appears to be the least likely
mechanism in explaining the reduction of these specific
enzyme activity levels. Differential mitochondrial transcriptional regulation is also unlikely since a common
promoter with co-ordinated transcription of mtDNA-encoded complex I, III, IV and V subunits is operative. Also,
unchanged levels of specific mitochondrial transcripts (e.g.
steady state level of ATPase 6 mRNA) were noted in
paced animals (data not shown). Studies directed to assess
the post-transcriptional regulation of mitochondrial enzymes in cardiac failure including translational, import and
assembly levels for these enzyme subunits are therefore
needed.
Increased levels of proinflammatory cytokines including
TNF-a, which have been demonstrated to play a role in the
pathogenesis of CHF [4], may be pivotally involved in the
mitochondrial dysfunction observed in the paced dogs.
Elevated levels of circulating TNF-a and its soluble
receptors have been reported in patients with CHF. The
rapid production of cardiac TNF-a in response to a variety
of physiological insults has supported its assignment as a
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J. Marın-Garcıa
‘cardiac stress response’ gene [31]. In transgenic mice with
cardiac-restricted overexpression of TNF-a, a heart failure
phenotype resulted characterized by left ventricular
dysfunction, elevated levels of TNF-a in the peripheral
circulation from cardiac spillover and biochemical markers
indicating high levels of oxidative stress [32]. Moreover,
mitochondrial dysfunction has been shown to be a key
element in the mechanism of TNF-a action in an assortment of cell types including skeletal muscle and cardiomyocytes [5–7]. Changes in mitochondrial membrane
permeability as well as the increased generation of reactive
oxygen species (ROS) primarily produced by mitochondrial electron transport complexes have been proposed to
be direct results of TNF-a action [5,6]. In addition,
mitochondrial respiratory activities are reduced in cardiomyocytes treated with TNF-a [7]. Studies with cells
treated with TNF-a have recently shown that the mitochondrial cytochromes are critical targets of TNF-a action;
cytochrome c is released to the cytosol through the
mitochondrial membrane pore, cytochrome b (a major
component of complex III) is reduced as are cytochromes
cc 1 and aa 3 [6]. Ceramide, another mediator of TNF-a
function, has been reported to selectively inhibit complex
III activity in isolated cardiac mitochondria [33]. The
TNF-a induced changes not only fundamentally modulate
mitochondrial respiration but also allow mitochondria to
play a key role in signalling redox and apoptotic changes
to the cell. Cytokines have also been shown to impact on
mitochondrial function during physiological stress conditions such as myocardial injury. Recent studies have
shown that cytokine-mediated increases in inducible nitric
oxide synthase (iNOS) directly inhibit mitochondrial respiration in cardiomyocytes [34,35]. The role of TNF-a in
signalling mitochondrial dysfunction in both cardiac and
skeletal muscle offers an attractive hypothesis which needs
to be further critically examined.
Our findings show an inverse correlation of TNF-a
levels with activity levels of complex III and V in paced
animals consistent with a potential contributory role of
TNF-a in the observed mitochondrial dysfunction. In
addition, the demonstration of markedly increased left
ventricular tissue aldehyde level in paced dogs indicates
increased levels of myocardial oxidative stress, further
evidence of free radical-induced damage in this model. The
role of NO-dependent pathways (another potential molecular mechanism for TNF-a to mediate mitochondrial
changes) appears to be less operative in this model since
paced dogs have recently been shown to have neither
increases in iNOS and eNOS activities nor in transcript
levels in left ventricular tissues [36]. In order to further
probe the potential relationship of TNF-a to specific
mitochondrial enzyme dysfunction during CHF, future
experiments to determine the precise timing of TNF-a
elevation and ROS changes relative to the onset of
mitochondrial enzymatic changes, as well as gauging the
effects on mitochondrial function by selective blocking of
109
TNF-a binding and function (at the level of its receptors),
will be useful.
Another indication of mitochondrial dysfunction,
myocardial mtDNA damage, was detectable in over 50%
of the paced animals as compared to controls. However,
given the low level of the mtDNA deletions found in this
study, mtDNA deletions are unlikely to be a causal factor
for either the reduced enzyme activity levels of complex
III and V, or a key mediator for progression of heart
failure. Previously, it have been suggested that increased
levels of mtDNA deletions are a result of increased oxygen
free radical-mediated damage to mtDNA with the generation of free radicals largely from hypoxic mitochondria
and from respiratory dysfunction [37]. This contention is
further supported by increased levels of specific mtDNA
deletions noted in canine cardiac tissues in response to
myocardial ischemia generated by ameroid constriction
[38].
In children with cardiomyopathy, the use of skeletal
muscle biopsies (instead of endomyocardial biopsies) has
been recommended to assess respiratory enzyme function
in the diagnostic evaluation of mitochondrial-mediated
cardiomyopathy [17]. Pronounced deficiencies in cytochrome c oxidase (complex IV) have been shown in both
cardiac and skeletal muscle of patients with Kearns-Sayre
syndrome and cardiomyopathy [39]. The findings of
mitochondrial dysfunction in skeletal muscle as well as in
cardiac tissue in this study suggest that there may exist a
commonality in the signaling for mitochondrial dysfunction in both skeletal muscle and cardiac tissues in heart
failure. While more evidence confirming these findings is
warranted, our current findings of skeletal muscle mitochondrial dysfunction in paced dogs provides further
support that changes in skeletal muscle may reflect
changes in cardiac muscle.
Currently, there is a gap in information about the
correlation between the physiological, biochemical and
molecular events in heart failure. There is a need for better
ways to quantify changes in mitochondrial bioenergetics,
structure and function not only in heart but also in skeletal
muscle. This would include an evaluation of the levels of
mitochondrial OXPHOS and respiratory enzyme activities,
mtDNA, ATP and NADH, as well as a comprehensive
investigation of the timing of mitochondrial changes as a
function of progression in the severity of cardiac failure.
Our findings suggest that reduction in specific mitochondrial enzyme activity levels and changes in mtDNA
integrity may have the potential of becoming sensitive
markers of tissue damage and progression of heart failure.
Acknowledgements
This work was supported in part by a grant-in-aid from
the Heart and Stroke Foundation of Ontario (Toronto,
Ontario). The authors acknowledge the expert technical
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J. Marın-Garcıa
assistance provided by Andrea DiRaddo and Marina
Romanova.
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