(Circulation. 1999;99:807-812.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Skeletal Muscle Mitochondrial DNA Injury in Patients With Unilateral Peripheral Arterial Disease
From the Department of Medicine, Harbor-UCLA Medical Center (H.K.B., E.P.B.), Torrance, Calif; University of Colorado Health Sciences Center (W.R.H.), Denver, Colo, and Cleveland VA Medical Center (C.L.H.), Departments of Pharmacology and Medicine, Case Western Reserve University, Cleveland, Ohio.
Correspondence to Eric P. Brass, MD, PhD, Department of Medicine, Harbor-UCLA Medical Center, 1000 W Carson St, Torrance, CA 90509. E-mail ebrass{at}ucla.edu
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Abstract |
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Background—Patients with peripheral arterial disease (PAD) have exercise limitation due to claudication-limited pain and metabolic alterations in skeletal muscle. PAD is also associated with oxidative stress, which is a known cause of mitochondrial DNA (mtDNA) injury. The present study was designed to test the hypothesis that PAD is associated with mtDNA injury, as reflected by an increased frequency of a specific 4977–base pair (bp) mtDNA deletion mutation.
Methods and Results—The deletion frequency was quantified in gastrocnemius muscle of 8 patients with unilateral PAD and 10 age-matched control subjects with the use of polymerase chain reaction methodologies. Muscle from the hemodynamically unaffected (less affected) PAD limb showed an 8-fold increased deletion frequency and the hemodynamically affected (worse affected) PAD limb had a 17-fold increased deletion frequency compared with muscle from control subjects. The frequency of the 4977-bp deletion in the worse-affected limb was positively correlated with the age of the patients but not the claudication-limited exercise performance of the patients. Total mtDNA content, citrate synthase activity, and cytochrome c oxidase activity were not different in the muscle from the 3 limb populations. However, the ratio of citrate synthase to cytochrome c oxidase was higher in the worse- versus less-affected limbs of PAD patients.
Conclusions—The present study demonstrates a large increase in the frequency of the mtDNA 4977-bp deletion in patients with PAD but in a distribution not limited to the hemodynamically affected limb.
Key Words: peripheral vascular disease • aging • metabolism
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Introduction |
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Peripheral arterial disease (PAD) results from atherosclerotic occlusion and impairment of arterial oxygen delivery to the lower limb1 and is associated with development of claudication pain with exercise.2 3 Although the initial insult in PAD is circulatory, peripheral hemodynamics are poor predictors of exercise capacity in PAD patients.4 Thus, factors intrinsic to the skeletal muscle may contribute to the functional impairment of patients with PAD.
Metabolic changes occur in the skeletal muscle of PAD patients.5 6 7 Muscle mitochondrial enzyme expression is altered,5 8 9 intermediates of oxidative metabolism accumulate,6 10 the kinetic response to exercise is slowed,11 and exercise training is abnormal12 13 in these patients. These metabolic features are similar to those in mitochondrial myopathies7 11 and suggest functionally relevant metabolic sequelae from the chronic hemodynamic abnormalities in PAD patients.
Somatic mutations to mitochondrial DNA (mtDNA) have been hypothesized to cause acquired mitochondrial dysfunction.14 15 mtDNA encodes for 13 polypeptides critical for electron transport chain function.14 16 Well-characterized mtDNA mutations arise from mutation hot spots.17 18 A 4977–base pair (bp) deletion spanning mtDNA nucleotide pairs 8469 to 13 447 (standard human mitochondrial DNA nomenclature used throughout; see Reference 16 ) has been reported frequently.14 19 20 21 Because each mitochondrion contains multiple copies of the genome,14 injury results in heteroplasmy in which each cell contains mtDNA molecules with varied sequences.14 22 Increased mtDNA mutation frequency occurs in humans with aging23 24 25 and in ischemic myocardium.19 21 The impact of acquired mtDNA injury in disease pathophysiology has been difficult to define owing to the lack of data relating mtDNA injury to mitochondrial and tissue function.
PAD provides a unique model for assessing the importance of mtDNA mutations in a clinically important disease. PAD is associated with oxidative stress26 27 analogous to the suggested mtDNA injury mechanism in ischemic heart.14 19 21 Patients with unilateral PAD permit specific influences of ischemia on mtDNA mutation frequency to be defined. Exercise testing and measurement of mitochondrial enzyme activities permits functional measurements as well. The present study was designed to test the hypothesis that PAD is associated with mtDNA injury, as reflected by an increased frequency of the specific 4977-bp mtDNA deletion.
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Methods |
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Patients
Eight patients with unilateral PAD and 10 age-matched, sedentary control subjects were recruited. A history and physical examination were performed, and subjects underwent assessment of ankle/brachial index (ABI; ratio of systolic blood pressures in ankle to arm).28 Unilateral PAD was identified as previously described.28 The limb with hemodynamically determined PAD is referred to as the "worse affected" limb, and the limb with ABI in the normal range is referred to as "less affected." All subjects underwent graded treadmill testing and peak oxygen uptake (
O2)
measurement.13 28 29 PAD subjects underwent needle
biopsies of each gastrocnemius muscle at rest as
described.6 13 Eight control subjects had a muscle sample
taken from 1 leg, and 2 control subjects had a muscle sample taken from
both legs. Samples were immediately frozen in liquid nitrogen and
stored at -80°C until analysis. All protocols were approved
by appropriate Institutional Review Boards, and subjects gave
informed consent.
4977-bp Deletion Frequency
Total cellular DNA was extracted from 10 to 20 mg of frozen
muscle by use of a QIAamp kit (Qiagen, Inc). Before polymerase
chain reaction (PCR) amplifications were performed, DNA samples were
digested with PstI (acting at nucleotide pairs
6910 and 9020) and HindIII (acting at nucleotide
pairs 6203, 11 680, and 12 567) (Promega, Inc) for 16 hours at 37°C
to linearize the mtDNA and cut the sequence within the 4977-bp deletion
region.19 The digested DNA was extracted with
phenol/chloroform, precipitated, and washed with ethanol, dissolved in
Tris-HCl, 10 mmol/L, pH 9.0, and remeasured before dilution and
use.
The proportion of total mtDNA containing a specific 4977-bp deletion
(deletion of 8469 to 13 447 nucleotide
pairs16 ) was determined with a serial dilution PCR method
modified from Corral-Debrinski et al.19 Two sets of PCR
reactions were performed on each sample. For the first amplification,
primers amplified a region not affected by the 4977-bp deletion or any
other known mtDNA deletions (5'-TTC AAA TTC CTC CCT GTA CG- 3' [primer
1], complementary to bases 3108 to 3127, and 5'-TTG GGC TAC TGC TCG
CAG TG-3' [primer 2], complementary to bases 3701 to 3720). This
amplification yields a 613-bp product from both wild-type and mtDNA
containing the 4977-bp deletion and is termed the total mtDNA
product. The second set of primers flanked the 4977-bp deletion and
yielded a 593-bp product only if the 4977-bp deletion was
present (5'- CTC TAG AGC CCA CTG TAA AGC TAA-3' [primer 3],
complementary to bases 8283 to 8305, and 5'-GTT GAG GTC TAG GGC TGT
TA-3' [primer 4], complementary to bases 13 832 to 13 851). PCR
amplifications (100 µL total volume) contained 200 µmol/L of
each dNTP, 1.5 mmol/L MgCl2, 10 mmol/L
Tris-HCl, and 50 mmol/L KCl, 0.3 µmol/L primers, and 5 U of
Taq polymerase (Boehringer Mannheim). For PCR amplification of
the 613-bp total mtDNA product, conditions were 2 minutes of
denaturation at 94°C (reduced to 1 minute in the second cycle and 30
seconds in 33 subsequent cycles), 2 minutes of annealing at 51°C, and
2 minutes of polymerization at 72°C. After 35 cycles, a 7-minute
extension at 72°C was performed. Amplification of the 593-bp deletion
product was conducted under the same conditions except that
annealing was performed at 56°C.19 Ethidium
bromide–stained 2% agarose size-fractionation gels were photographed,
and negative films were quantified with a Bio-Rad GS-700 imaging
densitometer (Bio-Rad Laboratories). Each gel included a DNA mass
ladder (Gibco BRL) in duplicate. The signal for the 800-bp fragment was
used as a standard and to normalize the signal for each PCR
product. For each sample, the PCR reactions were run at a minimum
of 6 different DNA template concentrations (typically varying over the
range of 2 to 200 ng per tube for the deletion amplification and 0.001
to 5 ng per tube for the total mtDNA amplification), which permitted
generation of a product optical density (OD) versus template
concentration curve. The ratio of the template DNAs required to yield
the same amount of product was used as a measure of the relative
amounts of deleted mtDNA versus total mtDNA in the sample (Figure 1
).
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=total DNA, 
=4977-bp deletion product). The ratio of the
template amounts required for the same yield was a measure of the
relative amounts of deleted mtDNA vs total mtDNA.This method demonstrated similar PCR amplification efficiencies for the deletion-specific and wild-type reactions, and no 593-bp deletion product was detected in DNA from young healthy subjects (<0.01% frequency). The coefficient of variation was found to be 20% when a single sample was run multiple times on separate days. The validated lower limit of detection was 0.01%, and thus all estimates that yielded deletion frequencies <0.01% were conservatively reported as 0.01%.
Total mtDNA Content
A competitive PCR strategy was used for the measurement of total
mtDNA.30 A 500-bp internal standard was generated with a
PCR-MIMIC construction kit from Clontech with a nonhomologous DNA
fragment of known sequence. The internal standard fragment was
generated with 2 rounds of PCR amplification reactions, the first with
composite primers and the second with mtDNA-specific primers. Composite
(40-mer) upstream and downstream primers were designed such that each
composite primer consisted of a 20-nucleotide base mtDNA
template-specific sequence, followed by a 20-nucleotide
base sequence complementary to the MIMIC DNA fragment (5'-TTC AAA TTC
CTC CCT GTA CGC GCA AGT GAA ATC TCC TCC G-3', composite upstream primer
5, and 5'-TTG GGC TAC TGC TCG CAG TGG GTT TAC ATA CTC TGG AGC A-3',
composite downstream primer 6). The PCR product generated was then
used in a second round of PCR amplification with mtDNA-specific primers
(primers 1 and 2 used for the amplification of the 613-bp total mtDNA
product, discussed above) to provide the internal standard.
Various amounts of internal standard DNA fragment (typically 6.0x101 to 6.0x105 molecules, including at least 6 different concentrations) were added to a known amount of total muscle DNA (typically 0.01 to 0.1 ng). PCR amplification was performed for 30 cycles with primers 1 and 2 under conditions for the amplification of the 613-bp mtDNA product as described above, except that the primer concentration was 0.6 µmol/L. This reaction thus yielded the 613-bp product from mtDNA and a 500-bp fragment from the competitor template. The PCR reaction products were separated on 2% agarose gel, negative films of the ethidium bromide stained gels were measured by densitometry, and the logs of the ratio of the mtDNA-specific product and internal standard ODs were plotted against the log of molecules of internal standard added to the reaction. We calculated the mtDNA content by determining the x intercept for the point at which the ratio of the 613- and 500-bp products was 1 (log 0).
Enzyme Assays
Muscle homogenates (10 mg/mL) of frozen tissue
prepared in 220 mmol/L mannitol, 70 mmol/L sucrose, 10
mmol/L MOPS, and 1% cholate (pH 7.0) were used for enzyme and protein
assays. Citrate synthase activity was determined by the method of
Srere,31 cytochrome c oxidase by the method of Wharton and
Tzagoloff,32 and noncollagenase protein by the
method of Lilienthal et al.33
Statistical Analysis
Data are expressed as mean±SEM. Student's t test
(paired) was used to compare data for the worse- and less-affected
limbs of PAD patients, and unpaired t tests were used for
comparison between PAD and control subjects. Analyses of the
4977-bp deletion frequency data were prospectively based on
log-transformed values to yield a normally distributed population. The
values for both legs were averaged before population analyses
were performed for control subjects biopsied in each leg.
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Results |
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All PAD and control subjects were male and of similar age (Table 1
). PAD patients had a peak oxygen
consumption only 50% that of the control subjects. The ABI of the
less-affected limb in the PAD subjects did not differ from controls,
whereas the worse-affected limb had an ABI <50% of the control value.
The PAD group included more smokers and as a group had a heavier
smoking history than the control subjects (22.5±6.9 pack-years and
44.2±8.1 pack-years in control and PAD subjects, respectively;
P<0.05).
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As expected, the gastrocnemius muscle from the control subjects showed
a low (0.05%) 4977-bp deletion frequency (Table 2). In contrast, the less-affected PAD
limbs showed an 8-fold higher deletion frequency than controls
(P<0.05), and the worse-affected PAD limbs demonstrated a
17-fold higher deletion frequency than control subjects
(P<0.01; Table 2). The frequency of the deletion was
not different in worse-affected limbs versus less-affected limbs in the
PAD population (P=0.119). Six of the 8 PAD subjects had
higher deletion frequencies in the worse- versus less-affected limbs
(Figure 2). Significant overlap was seen
in the deletion frequencies in the worse- and less-affected limbs among
the 8 patients (Figure 2). The deletion frequencies in the
patients' less-affected and worse-affected limbs were strongly
correlated (r=0.88, P<0.01).
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The frequency of the 4977-bp deletion in the worse-affected limbs from
the PAD population was positively correlated with age (Figure 3). A similar correlation of lesser slope
was seen in the less-affected limb (log deletion
frequency=0.0468xage-5.78; r=0.735). The low
deletion frequency in the control muscles precluded a meaningful
correlation analysis in this population. There were no
significant relationships between the 4977-bp deletion frequency in the
worse-affected limbs and either the patients' ABI or
claudication-limited exercise performance.
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Muscle mitochondrial expression is closely linked to total mtDNA
content.34 35 No significant differences in the total
mtDNA content were found between control subjects and the less- or
worse-affected limbs of PAD patients (Table 2
). No differences
in the activity of citrate synthase or cytochrome c oxidase were
detected in control subjects versus PAD-affected limbs or between the
less- or worse-affected limbs of PAD patients (Table 3). However, the ratio of citrate
synthase to cytochrome c oxidase was increased in muscle from the
worse-affected PAD limbs compared with the least-affected limbs
(P<0.05).
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Discussion |
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Although PAD is a consequence of atherosclerosis-induced blood flow limitations, lower extremity hemodynamics do not predict peak exercise performance.3 4 12 Metabolic changes have been defined in skeletal muscle of PAD patients with many features similar to those in mitochondrial myopathies.5 6 7 8 10 11 12 13 The molecular basis for altered muscle metabolism in PAD is unknown. The current study demonstrates that the frequency of the 4977-bp mtDNA deletion is increased in PAD but is not specific for the ischemic limb.
The 17-fold increase in frequency of the 4977-bp deletion in the
worse-affected limb of the PAD subjects compared with controls is
consistent with the increased deletion frequency seen in
ischemic myocardium22 24 and the
preliminary observations of Egawhary et al36 in nonmuscle
cells in PAD. The most severely affected individual demonstrated a
deletion frequency 88-fold higher than the control group mean (Figure 2) and 440-fold higher than the individuals at the low end of
the control group range (data not shown). The 4977-bp deletion arises
from alternate pairing during mitochondrial genome
replication,17 and the frequency of this deletion is
increased in a variety of settings, including enhanced free radical
generation.24 37 Ischemia and
ischemia/reperfusion are associated with increased free radical
generation from the mitochondrial electron transport
chain,38 39 and PAD is associated with a state of
increased oxidative stress.26 27
There was a strong trend for a higher deletion frequency in the worse-
versus less-affected limb in individual patients (2-fold higher
absolute mean deletion frequency, P=0.119, with 6 of 8
paired limbs demonstrating the relationship; Figure 2). The
current study was not powered to detect differences in deletion
frequency of this small magnitude (a total of 
20 patients would be
required), and thus the lack of statistical significance may be
misleading. Nonetheless, the major increase in mtDNA injury appears to
be independent of local ischemia in the PAD population. Smoking
is prevalent in the PAD population and has been hypothesized to
accelerate mtDNA injury.40 However, the control group
contained smokers, and no difference in 4977-bp deletion frequency
within the control group as a function of smoking was identified (data
not shown). Alternatively, mtDNA injury in the less-affected limb may
reflect subclinical arterial disease that is sufficient to
induce injury mechanisms despite the lack of symptoms and ABIs in the
normal range. This is unlikely given the strong relationship between
the degree of mtDNA injury in the less-affected limb and the injury in
the worse-affected muscle (Figure 2). Thus, the oxidative stress
associated with PAD26 27 appears to induce mtDNA damage in
a distribution beyond the ischemic vascular bed, which may be
further aggravated by ischemia in the affected limb.
Accumulation of mtDNA mutations occurs at a slow rate in normal
aging.25 PAD may be viewed as accelerating this process.
In the PAD limb, the frequency of the 4977-bp deletion was correlated
with the patient's age (Figure 3). The 4977-bp deletion is only
one of many deletion and point somatic mutations that have been
identified in human mtDNA. Thus, the quantitative assessment of mtDNA
injury demonstrated here does not reflect the full extent of mtDNA
damage but rather the lower limit of injury and the relative degree of
injury between samples.
Somatic mtDNA mutations have been associated with a number of
degenerative diseases.14 15 However, the causative role of
the mtDNA injury in disease pathophysiology has not been well
established in any clinical model. The patient with unilateral PAD
illustrates the difficulty in interpreting mtDNA injury as a mechanism
in disease pathophysiology. Patients with unilateral PAD clearly
lateralize symptoms, hemodynamics, and biochemical
abnormalities between the 2 limbs.6 Assessment of mtDNA
injury in only the diseased limb might have suggested that mtDNA injury
was relevant to the pathogenesis of the myopathy of PAD. However,
examination of the less-affected limb makes this hypothesis less
tenable. The high degree of mtDNA injury in the less-affected limb of
some subjects and the overlap in deletion frequency in the less- versus
worse-affected limb population (Figure 2) make a primary role
for mtDNA injury in the disease unlikely. The deletion frequency in the
worse-affected limb was not correlated with either the patient's
hemodynamics (ABI) or claudication-limited function.
Similarly, cytochrome c oxidase activity was not decreased in the
worse-affected limb of the PAD patients compared with either control or
the less-affected limb (Table 3).
Although mitochondrial content varies in muscle with training and
detraining, the relative amounts of each of the mitochondrial
constituents remains constant.35 41 Citrate synthase
activity is increased in PAD-affected muscle,5 13 and this
trend is also seen in Table 3. In mitochondrial myopathies,
expression of nuclear-encoded mitochondrial enzymes is often increased
compared with mtDNA-encoded enzymes.42 Consistent
with this concept in PAD patients, the ratio of citrate synthase
(nuclear encoded) to cytochrome c oxidase (dependent on mtDNA-encoded
subunits) activities was increased in the worse- versus less-affected
limb. Again, the present study was not powered to detect
differences in this ratio between the control and PAD limbs due to the
greater intersubject versus intrasubject variability.
In summary, PAD is associated with accumulation of mtDNA injury, as evidenced by the high 4977-bp mtDNA deletion frequency, but this injury is not limited to the worse-affected limb in patients with unilateral disease. mtDNA injury in the less-affected limb and the relative preservation of mtDNA content and cytochrome c oxidase activity make it unlikely that a simple model of mtDNA injury causing decreased expression is responsible for the metabolic derangements seen in PAD.
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Acknowledgments |
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This study was supported in part by the Harbor-UCLA Research and Education Institute, Sandoz Gerontological Foundation, Sigma Tau Pharmaceuticals, NIH Award in Vascular Medicine (NIH HL-02825), and the American Heart Association, Greater Los Angeles Affiliate, Los Angeles, Calif. The authors thank Kalpana Patel and Timothy Bauer for their technical help.
Received May 27, 1998; revision received September 4, 1998; accepted September 25, 1998.
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42. Hoppel CL, Kerr DS, Dahms B, Roessmann U. Deficiency of the reduced nicotinamide adenine dinucleotide dehydrogenase component of complex I of mitochondrial electron transport. J Clin Invest. 1987;80:71–77.The hypothesis that peripheral arterial disease (PAD) is associated with mitochondrial DNA (mtDNA) injury, as reflected by an increased frequency of a specific 4977-bp mtDNA deletion mutation, was tested in patients with unilateral PAD. Muscle from the hemodynamically affected PAD limb had a 17-fold increased deletion frequency versus controls, which was positively correlated with the age of the patients. The hemodynamically unaffected limb had an 8-fold increased deletion frequency versus controls. The study demonstrates a large increase in the mtDNA 4977-bp deletion frequency in PAD but in a distribution not limited to the hemodynamically affected limb.
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 E. P Brass, W. R Hiatt, and S. Green Skeletal muscle metabolic changes in peripheral arterial disease contribute to exercise intolerance: a point-counterpoint discussion Vascular Medicine, November 1, 2004; 9(4): 293 - 301. [Abstract] [PDF]
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 L.-P. Lai, C.-C. Tsai, M.-J. Su, J.-L. Lin, Y.-S. Chen, Y.-Z. Tseng, and S. K. S. Huang Atrial Fibrillation Is Associated With Accumulation of Aging-Related Common Type Mitochondrial DNA Deletion Mutation in Human Atrial Tissue Chest, February 1, 2003; 123(2): 539 - 544. [Abstract] [Full Text] [PDF]
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 K. Yamagata, K. Muro, J. Usui, M. Hagiwara, H. Kai, Y. Arakawa, Y. Shimizu, C. Tomida, K. Hirayama, M. Kobayashi, et al. Mitochondrial DNA Mutations in Focal Segmental Glomerulosclerosis Lesions J. Am. Soc. Nephrol., July 1, 2002; 13(7): 1816 - 1823. [Abstract] [Full Text] [PDF]
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 P. Cerretelli and B. Grassi Gas Exchange, MRS and NIRS Assessment of Metabolic Transients in Skeletal Muscle Integr. Comp. Biol., April 1, 2001; 41(2): 229 - 246. [Abstract] [Full Text] [PDF]
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 E. P. Brass, W. R. Hiatt, A. W. Gardner, and C. L. Hoppel Decreased NADH dehydrogenase and ubiquinol-cytochrome c oxidoreductase in peripheral arterial disease Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H603 - H609. [Abstract] [Full Text] [PDF]
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E. P Brass, H. Wang, and W. R Hiatt Multiple skeletal muscle mitochondrial DNA deletions in patients with unilateral peripheral arterial disease Vascular Medicine, November 1, 2000; 5(4): 225 - 230. [Abstract] [PDF]
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E. P Brass and W. R Hiatt Acquired skeletal muscle metabolic myopathy in atherosclerotic peripheral arterial disease Vascular Medicine, February 1, 2000; 5(1): 55 - 59. [Abstract] [PDF]
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 T. A. Bauer, J. G. Regensteiner, E. P. Brass, and W. R. Hiatt Oxygen uptake kinetics during exercise are slowed in patients with peripheral arterial disease J Appl Physiol, August 1, 1999; 87(2): 809 - 816. [Abstract] [Full Text] [PDF]
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