Exercise Physiology

Exercise Physiology
Antioxidative Effects of Exercise Training in Patients With
Chronic Heart Failure
Increase in Radical Scavenger Enzyme Activity in Skeletal Muscle
Axel Linke, MD; Volker Adams, PhD; Paul Christian Schulze, MD; Sandra Erbs, MD;
Stephan Gielen, MD; Eduard Fiehn, MD; Sven Möbius-Winkler, MD; Andreas Schubert, PhD;
Gerhard Schuler, MD; Rainer Hambrecht, MD
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Background—In chronic heart failure (CHF), cross-talk between inflammatory activation and oxidative stress has been
anticipated in skeletal muscle (SM). The role of the radical scavenger enzymes superoxide dismutase (SOD), catalase
(Cat), and glutathione peroxidase (GPX), which remove oxygen radicals, has never been assessed in the SM in this
context. Moreover, it remains unknown whether exercise training augments the activity of these enzymes in CHF.
Methods and Results—Twenty-three patients with CHF were randomized to either 6 months of exercise training (T) or a
sedentary lifestyle (C); 12 age-matched healthy subjects (HS) were studied in parallel. Activity of Cat, SOD, and GPX
was assessed in SM biopsies before and after 6 months (6 months). Oxidative stress was determined by measuring
nitrotyrosine formation. SOD, Cat, and GPX activity was reduced by 31%, 57%, and 51%, respectively, whereas
nitrotyrosine formation was increased by 107% in SM in CHF (P⬍0.05 versus HS). In CHF, exercise training
augmented GPX and Cat activity in SM by 41% (P⬍0.05 versus before and group C) and 42% (P⬍0.05 versus before
and group C), respectively, and decreased nitrotyrosine production by 35% (from 3.8⫾0.4% tissue area before to
2.5⫾0.3% after 6 months; P⬍0.05 versus before).
Conclusions—The reduced activity of major antioxidative enzymes in the SM of CHF patients is associated with increased
local oxidative stress. Exercise training exerts antioxidative effects in the SM in CHF, in particular, due to an
augmentation in activity of radical scavenger enzymes. (Circulation. 2005;111:1763-1770.)
Key Words: exercise 䡲 heart failure 䡲 muscles 䡲 free radicals 䡲 enzymes
I
n patients with chronic heart failure (CHF), excessive
oxidative stress in skeletal muscle (SM) has been linked to
peripheral hypoperfusion as a consequence of low cardiac
output and peripheral endothelial dysfunction.1– 4 On one
hand, reactive oxygen species (ROS), produced by a variety
of enzymes,1,5– 8 are known to induce the expression of
inflammatory cytokines, eg, in the SM of patients with CHF.9
On the other hand, cytokines themselves might promote the
production of reactive oxygen metabolites.9,10 These findings
emphasize the cross-talk between local inflammation and
oxidative stress.9 –12 Excessive production of free radicals
partially contributes to the impairment of muscle function and
apoptosis of SM fibers, with the consequent loss in muscle
bulk.1,13–16 Nevertheless, SM contains an enzymatic antioxidative system encompassing copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPX), and catalase (Cat),
which protect the cells from attacks by ROS.17 Currently, it is
unknown whether this enzyme system adapts to the enhanced
production of ROS or whether the impaired activity of radical
scavenger enzymes leads to a further increase in local
oxidative stress. Therefore, we aimed to determine whether
alteration in the activity of total SOD, GPX, and Cat is
associated with enhanced production of free radicals in the
SM of patients with CHF. Moreover, we were interested to
determine whether significant cross-talk exists between local
inflammation and the activity of key radical scavenger
enzymes.
Exercise training has been established as adjuvant therapy
in CHF, which improves exercise capacity and partially
reverses intrinsic alterations of SM.3,4,18,19 Most intriguing
was the training-induced reduction of the local expression of
tumor necrosis factor (TNF)-␣ that is known to diminish
intracellular levels of reduced glutathione (ie, glutathione
containing an SH group [GSH]), which is an essential
cofactor of GPX and hence, decrease GPX activity11,20,21;
however, it is currently unknown whether the trainingmediated downregulation of SM inflammation is associated
Received August 6, 2004; revision received December 29, 2004; accepted January 6, 2005.
From the Heart Center, Departments of Cardiology and Cardiac Surgery (A.S.), University of Leipzig, Leipzig, Germany.
The online-only Data Supplement can be found with this article at http://www.circulationaha.org.
Correspondence to Axel Linke, MD, Department of Internal Medicine/Cardiology, Heart Center, University of Leipzig, Strümpellstrasse 39, 04289
Leipzig, Germany. E-mail [email protected]
© 2005 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000165503.08661.E5
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April 12, 2005
with restoration of the local enzymatic radical scavenger
system. Therefore, we aimed to investigate whether aerobic
exercise training in stable CHF reconstitutes the activity of
SOD, Cat, and GPX, thereby reducing oxidative stress and
apoptosis of myocytes in SM.
Methods
A detailed description of the methodology is provided in the online
Data Supplement.
TABLE 1. Baseline Characteristics of Patients With CHF and
Sedentary HSs
Sedentary HSs
(n⫽12)
Age, y
V̇O2max, mL䡠kg⫺1䡠min⫺1
LVEF, %
DCM/IHD, n/n
Patient Selection
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The Ethics Committee of the University of Leipzig approved the
protocol of this study, and written, informed consent was obtained
from all patients and healthy subjects (HSs). Patients with CHF were
randomized to either 6 months of exercise training (training group,
T) or a sedentary lifestyle (control group, C). A total of 12
age-matched men, who were admitted for nonspecific chest pain to
rule out coronary artery disease, served as HSs. Inclusion and
exclusion criteria for CHF patients are described in detail in the
online Data Supplement.
Training Protocol
During the first 2 weeks, patients in group T exercised in hospital 4
to 6 times daily for 10 minutes each on a bicycle ergometer adjusted
to a workload at which 70% of peak oxygen uptake was reached. The
target heart rate for home training was defined as the heart rate
reached at 70% of maximum oxygen uptake. Patients were provided
with bicycle ergometers for home exercise training. They were
encouraged to exercise close to their target heart rate daily for 20
minutes for a period of 6 months and were expected to participate in
one group training session, consisting of walking, noncompetitive
ball games, and calisthenics, for 60 minutes each week. Patients
assigned to group C continued their sedentary lifestyle.
Exercise Testing, Respiratory Variables, and
SM Biopsy
CHF Patients,
Group T
(n⫽12)
CHF Patients,
Group C
(n⫽11)
56⫾4
55⫾2
52⫾3
26.4⫾1.7
19.0⫾0.8*
17.5⫾1.5*
70⫾2
26⫾3*
27⫾3*
䡠䡠䡠
9/3
7/4
NYHA, No. in class II/III
0/10/2/0
0/10/1/0
䡠䡠䡠
V̇O2max indicates maximal oxygen uptake; LVEF, left ventricular ejection
fraction; DCM, dilated cardiomyopathy; IHD, ischemic heart disease; and NYHA,
New York Heart Association.
*P⬍0.05 vs sedentary HSs.
photometrically with a commercially available kit (Immundiagnostik
AG).26
Measurement of Cytokine Expression and
Nitrotyrosine Formation
IL-1␤ and TNF-␣ expression was measured immunohistochemically.10 Nitrotyrosine formation was assessed as a second marker of
oxidative stress,27 representing a readout of peroxynitrite production
with a specific antibody.11 Myocytes were recognized by anti-SM
actin antibody staining; endothelial cells were identified by an
anti–von Willebrand Factor (vWF) staining.
Measurement of Cytokines in Serum
In patients with CHF and HSs, serum concentrations of TNF-␣ and
IL-1␤ were assessed with commercially available, highly sensitive
ELISA kits (R&D Systems).
Detection of Apoptosis
Exercise testing was performed on a calibrated, electronically braked
bicycle in an upright position. Respiratory gas exchange data were
determined continuously throughout the exercise test.22 Percutaneous needle biopsy samples were obtained from the vastus lateralis
muscle as described in the online Data Supplement.
Apoptosis was determined by use of the Apo Tag peroxidase
oligoligation apoptosis detection kit according to the instructions of
the manufacture (Chemicon). The number of total SM myocyte
nuclei and of apoptotic nuclei was counted, and the data are
expressed as number of apoptotic myocyte nuclei per 10 000 SM
myocyte nuclei.
Measurement of mRNA
Statistical Analysis
MnSOD, CuZnSOD, GPX, Cat, interleukin (IL)-1␤, and TNF-␣
mRNAs were quantified by real-time polymerase chain reaction with
specific primers (Light Cycler system, Roche Diagnostics Inc).11 The
mRNA of the radical scavenger enzymes and the cytokines was
expressed as a ratio to 18S rRNA, which was amplified as a
housekeeping gene.11
Quantification of Protein Expression
Protein expression of MnSOD, CuZnSOD, and Cat was determined
by Western blot techniques. Expression was quantified by applying
a 1-D analysis software package and was normalized to an internal
standard that was loaded onto each gel.
Quantification of Enzymatic Activities
The frozen biopsy samples were homogenized, and protein content
was determined.5 Activities of total SOD,23 GPX,24 and Cat25 were
measured photometrically according to standard protocols and expressed as units per milligram protein or milliunits per milligram
protein.
Measurement of Lipid Peroxidation as a Marker
of Oxidative Stress
Homogenates of SM were processed according to the instructions of
the manufacture, and lipid peroxide concentrations were determined
Mean⫾SE values were calculated for all variables. Intergroup and
intragroup comparisons were performed with a 2-way, repeatedmeasures ANOVA followed by the Tukey post-hoc test, a MannWhitney U test, or a Wilcoxon signed-rank test when appropriate. A
probability value ⬍0.05 was considered statistically significant.
Results
Baseline Characteristics and Clinical Follow-Up
A total of 23 CHF patients and 12 healthy, age-matched
subjects (Table 1) were enrolled in the study. Twelve CHF
patients were randomized to the training group, whereas 11
CHF patients remained sedentary as a control group. At
baseline, patients in groups T and C did not significantly
differ with respect to left ventricular ejection fraction, peak
oxygen uptake, and New York Heart Association class.
Age-matched HSs had normal parameters of cardiac function
(Table 1) and a higher maximal oxygen uptake (V̇O2max)
compared with CHF patients (18.3⫾0.8 mL · kg⫺1 · min⫺1 in
CHF versus 26.4⫾1.7 in HSs, P⬍0.001).
Medical therapy was similar in T and C groups and did not
change during the study period. Patients received angioten-
Linke et al
TABLE 2. mRNA Abundance and Protein Expression of Radical
Scavenger Enzymes in SM of Sedentary HSs and Patients
With CHF
Sedentary HSs
MnSOD
CuZnSOD
Radical Scavenger Enzyme Activity in CHF
TABLE 3. mRNA Abundance and Protein Expression of
Inflammatory Cytokines in SM of Sedentary HSs and Patients
With CHF
CHF Patients
Sedentary HSs
CHF Patients
0.91⫾0.19
2.15⫾0.17*
1.80⫾0.26†
mRNA
Protein
mRNA
Protein
TNF-␣, serum
2.69⫾0.73
2.08⫾0.30
1.28⫾0.27*
0.79⫾0.13†
TNF-␣ mRNA
0.30⫾0.09
TNF-␣ protein
0.76⫾0.10
1.40⫾0.06†
IL-1␤, serum
0.26⫾0.14
0.63⫾0.20
IL-1␤ mRNA
0.29⫾0.11
2.78⫾0.40‡
IL-1␤ protein
0.67⫾0.07
1.09⫾0.08†
0.31⫾0.08
1.90⫾0.18
0.16⫾0.03*
1.70⫾0.22
GPX
0.26⫾0.06
ND
0.06⫾0.01†
ND
Cat
92⫾13
1.03⫾0.09
45⫾6†
0.58⫾0.05†
Data are expressed in arbitrary units (mean⫾SEM).
ND indicates not determined.
*P⬍0.05, †P⬍0.001 vs sedentary HSs.
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sin-converting enzyme inhibitors (12 patients in group T and
9 patients in group C), digitalis (7 in T and 8 in C), diuretics
(8 in T and 8 in C), ␤-blockers (5 in T and 5 in C), nitrates
(1 in T and 2 in C), and statins (0 in T and 2 in C).
One patient with CHF in each group withdrew his consent
for study participation. The medication and baseline characteristics of those patients did not differ from those who
successfully participated in the entire study. No death or
cardiac decompensation occurred, and none of the patients
was admitted to the hospital during the study period.
The exercise training program effectively increased peak
oxygen uptake in group T by 25%, from 19.0⫾0.8 before to
23.7⫾1.4 mL · kg⫺1 · min⫺1 at 6 months (P⬍0.05 versus
before and group C), whereas V̇O2max remained unchanged
in group C (17.5⫾1.5 before to 17.8⫾1.2 mL · kg⫺1 · min⫺1
at 6 months).
Expression and Activity of Radical Scavenger
Enzymes in SM
Superoxide Dismutase
In patients with CHF, SM had a 31% lower total SOD activity
compared with that in HSs (5.7⫾0.4 U/mg in CHF versus
8.3⫾1.4 in HSs, P⬍0.05). MnSOD expression was diminished by 62% at the protein level (P⬍0.001 versus HSs) and
by 52% at the RNA level (P⬍0.05 versus HSs) in comparison
with HSs (Table 2). The CuZnSOD protein content was not
significantly attenuated (P⫽NS) in CHF patients, whereas
CuZnSOD mRNA was significantly reduced by 48%
(P⬍0.05 versus HS) compared with HSs (Table 2).
Catalase
In CHF, SM was characterized by a 57% reduction in Cat
activity (12.7⫾1.4 U/mg in CHF versus 29.5⫾4.7 in HSs,
P⬍0.001), in Cat protein content by 44% (P⬍0.001 versus
HS), and in Cat mRNA expression by 51% (P⬍0.001 versus
HSs) in comparison with those of HSs (Table 2).
Glutathione Peroxidase
In patients with CHF, the activity of GPX in SM was
significantly diminished by 51% (35.5⫾3.3 mU/mg in CHF
versus 71.9⫾9.6 g in HSs, P⬍0.001) compared with that in
HSs. This decrease in GPX activity was accompanied by a
77% attenuation of GPX mRNA expression (P⫽0.001 versus
HSs) in CHF (Table 2). To elucidate the underlying mechanism, we aimed to determine GPX protein expression. Un-
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Data are expressed as mean⫾SEM. Serum concentration are reported in
pg/mL. TNF-␣ and IL-1␤ mRNA expression were normalized to the expression
of 18S rRNA and were reported in arbitrary units. TNF-␣ and IL-1␤ protein
expression is reported in percentage of tissue area that stained positive for
respective cytokines.
*P⬍0.05, †P⬍0.01, ‡P⬍0.001, vs sedentary HSs.
fortunately, we (like others) discovered that all of the commercially available antibodies did not reveal signals on
Western blots because either cross-reactivity with human
proteins was missing or the size of the band was not the
expected one for GPX.28
Local Oxidative Stress
GPX and Cat are mainly detoxifying hydroxyl radicals that
would react with lipids from the SM. Those lipid peroxides
can be considered a measure of local oxidative stress and
partially represent a readout of GPX as well as Cat activity.
Local oxidative stress, measured as lipid peroxidation, in the
SM of patients with CHF was significantly elevated (by
151%) compared with HSs (377⫾70 ␮mol/mg protein in
CHF versus 150⫾50 in HSs, P⬍0.05). In addition, nitrotyrosine production, reflecting formation of the highly reactive
peroxynitrite radical,27 was significantly increased (by 103%)
in the SM of patients with CHF in comparison with HSs
(3.79⫾0.34% positive tissue area in CHF versus 1.87⫾0.45%
in HSs, P⬍0.05).
Circulating Cytokines, Local Cytokine Expression,
and Cross-Talk Between Local Inflammation and
Radical Scavenger Enzymes
TNF-␣ serum levels were significantly elevated (by 135%) in
CHF. On the contrary, IL-1␤ serum levels did not differ
between patients with CHF and HSs (Table 3). In CHF,
TNF-␣ and IL-1␤ mRNA expression in the SM was significantly elevated (6-fold and 9.5-fold, respectively), which was
associated with an increase in TNF-␣ and IL-1␤ protein
levels (by 84% and 63%, respectively) in comparison with
HSs (Table 3). Those cytokines were most likely primarily
myocyte derived because the majority of the tissue was found
to be composed of myocytes (Figure 1). In addition, there was
no correlation between serum levels of TNF-␣ and IL-1␤ and
local expression of the respective cytokines in SM. In
contrast, local TNF-␣ expression was inversely related to
GPX (r⫽⫺0.63, P⬍0.05) and Cat activity (r⫽⫺0.55,
P⬍0.05) in SM.
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Figure 1. A, Representative tissue section of SM biopsy sample from
patient with CHF (magnification 40-fold). B, Higher magnification (400fold) of area enclosed by white rectangle in A. Vessel layered by endothelial cells, that express vWF and are embedded in connective
tissue and surrounded by myocytes, is shown. Expression of SM
actin (red fluorescence) and vWF (green fluorescence) and staining of
nuclei with Hoechst dye (blue fluorescence) are shown.
Association Between Oxidative Stress and
Structural Alterations of SM
Apoptosis was found to occur more frequently in SM myocytes of patients with CHF (49⫾4 apoptotic nuclei/10 000
SM myocyte nuclei) compared with HSs (22⫾4 apoptotic
nuclei/10 000 SM myocyte nuclei, P⬍0.01). The functional
association between the increase in oxidative stress as measured by nitrotyrosine formation and the occurrence of
apoptotic cell death is supported by the close correlation
between both parameters (r⫽0.62, P⬍0.01).
Effects of Exercise Training on Activity of Radical
Scavenger Enzymes in Patients With CHF
Superoxide Dismutase
Regular physical exercise training failed to affect total SOD
activity (5.6⫾0.6 U/mg before versus 5.8⫾0.8 at 6 months,
Figure 2A), mRNA content (Table 4), or protein expression
(Table 5) of the 2 isoforms. In patients in group C, no changes
with respect to SOD mRNA content (Table 4), protein
expression (Table 5), and activity were determined (5.9⫾0.3
U/mg before versus 6.3⫾0.8 at 6 months).
Figure 2. A, SOD activity in vastus lateralis muscle of HSs and
patients of CHF groups C and T. B, Cat activity in vastus lateralis muscle of HSs and in patients of CHF groups C and T. Open
bars represent at beginning (B) and filled bars at end of 6
months. *P⬍0.05 vs HSs; †P⬍0.05 vs CHF group T at B;
‡P⬍0.05 vs CHF group C at 6 months.
Catalase
In CHF patients (group T), exercise training resulted in an
increase in Cat activity by 42% (from 12.4⫾2.1 U/mg before
to 17.6⫾2.7 at 6 months, P⬍0.05 versus before and change
versus group C; Figure 2B) without altering mRNA and
protein expression in SM (Tables 4 and 5). Despite the
improvement, Cat activity still tended to be low after 6
months of exercise training in CHF patients (17.6⫾2.7 U/mg
in CHF versus 29.5⫾4.7 U/mg in HSs, P⫽0.051). Cat
activity (Figure 2B), mRNA content, and protein expression
remained unchanged in group C (Tables 4 and 5).
TABLE 4. mRNA Abundance of Radical Scavenger Enzymes in
SM of Patients With CHF
CHF Patients, Group T
CHF Patients, Group C
Begin
Begin
6 Mo
6 Mo
MnSOD
1.28⫾0.32
1.47⫾0.27
1.27⫾0.45
1.41⫾0.33
CuZnSOD
0.15⫾0.05
0.14⫾0.07
0.17⫾0.05
0.17⫾0.07
GPX
0.05⫾0.02
0.06⫾0.02
0.07⫾0.02
0.06⫾0.02
Cat
45⫾9
49⫾8
45⫾8
43⫾9
Data are expressed in arbitrary units (mean⫾SEM).
Linke et al
TABLE 5. Protein Expression of Radical Scavenger Enzymes in
SM of Patients With CHF
CHF Patients, Group T
Radical Scavenger Enzyme Activity in CHF
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TABLE 6. Serum Concentration and mRNA Abundance of
Inflammatory Cytokines in SM of Patients With CHF
CHF Patients, Group C
CHF Patients, Group T
CHF Patients, Group C
Begin
6 Mo
Begin
6 Mo
Begin
6 Mo
Begin
6 Mo
MnSOD
0.88⫾0.20
0.97⫾0.19
0.68⫾0.16
0.63⫾0.18
TNF-␣ serum
2.24⫾0.21
2.10⫾0.31
2.05⫾0.26
2.47⫾0.61
CuZnSOD
1.70⫾0.31
1.73⫾0.23
1.69⫾0.33
1.73⫾0.34
TNF-␣ mRNA
1.71⫾0.39
0.92⫾0.19*
1.86⫾0.38
2.29⫾0.60
Cat
0.63⫾0.09
0.73⫾0.06
0.52⫾0.03
0.59⫾0.10
TNF-␣ protein
1.43⫾0.09
1.04⫾0.11†
1.37⫾0.09
1.46⫾0.10
IL-1␤, serum
0.59⫾0.26
0.42⫾0.18
0.67⫾0.32
0.48⫾0.17
IL-1␤ mRNA
2.79⫾0.73
1.81⫾0.67*
2.78⫾0.37
3.38⫾0.46
IL-1␤ protein
1.12⫾0.10
0.78⫾0.07†
1.06⫾0.13
1.26⫾0.13
Data are expressed in arbitrary units (mean⫾SEM).
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GPX Expression
Exercise training augmented GPX activity in SM by 41%
(from 35.3⫾4.3 mU/mg before to 49.9⫾3.5 at 6 months,
P⬍0.05 versus before and versus group C; Figure 3A) in
CHF patients, but the activity of this enzyme still tended to be
low when compared with that in HSs (49.9⫾3.5 mU/mg in
CHF versus 71.9⫾9.6 in HSs, P⫽0.052); however, GPX
mRNA expression was not altered by exercise training (Table
4). GPX activity (Figure 3A) and GPX mRNA expression did
not change during the study in group C (Table 4).
Data are expressed as mean⫾SEM. Serum concentration are reported in
pg/mL. TNF-␣ and IL-1␤ mRNA expression were normalized to the expression
of 18S rRNA and are reported in arbitrary units. TNF-␣ and IL-1␤ protein
expression is reported in percentage of tissue area that stained positive for the
respective cytokines.
*P⬍0.05 for the change vs CHF group C.
†P⬍0.01 vs at the beginning of the study.
Local Oxidative Stress
In CHF patients, local oxidative stress in SM was considerably reduced by 6 months of regular physical activity (decrease in lipid peroxidation by 57%, from 368⫾92 ␮mol/mg
protein before to 166⫾51 at 6 months, P⬍0.05 versus before)
but remained constantly elevated in the inactive control group
(386⫾100 ␮mol/mg protein before versus 398⫾124 at 6
months, Figure 3B). In parallel, nitrotyrosine production
decreased by 34% (from 3.76⫾0.36% positive tissue area
before to 2.48⫾0.30% at 6 months, P⬍0.05 versus before)
but tended to increase in group C (from 3.61⫾0.61% positive
tissue area before to 4.50⫾0.50% at 6 months, P⫽0.15 versus
before).
Circulating Cytokines, Local Cytokine Expression,
and Cross-Talk Between Local Inflammation and
Radical Scavenger Enzymes
During the study period, serum levels of TNF-␣ as well as
IL-1␤ did not change in either the CHF control or CHF
training group (Table 6). In contrast, exercise training resulted in a reduction in local TNF-␣ and IL-1␤ mRNA
expression (by a respective 46% and 35%) that was linked to
a decrease in TNF-␣ and IL-1␤ protein levels by 27% and
30%, respectively, in SM in CHF (Table 6). The decrease in
local TNF-␣ protein levels in the CHF training group was
associated with an enhancement of GPX (r⫽⫺0.68, P⬍0.05)
and Cat (r⫽⫺0.60, P⬍0.05) activity.
Association Between Oxidative Stress and
Structural Alterations of SM
Figure 3. A, GPX activity in vastus lateralis muscle of HSs and
patients of CHF groups C and T. Open bars represent at beginning (B) and filled bars at end of 6 months. *P⬍0.05 vs HS;
†P⬍0.05 vs CHF group T at B; ‡P⬍0.05 vs CHF group C at 6
months. B, Lipid peroxide concentrations in vastus lateralis
muscle of HSs and patients of CHF groups C and T. Open bars
represent at beginning (B) and filled bars at end of 6 months.
*P⬍0.05 vs HSs; †P⬍0.05 vs CHF group T at B.
Exercise training led to a reduction in apoptosis by 24% in
group T (from 49⫾6 apoptotic nuclei/10 000 SM myocyte
nuclei before to 37⫾8/10 000 SM at 6 months, P⬍0.05
versus before and for the change versus group C), whereas no
changes were observed in group C (from 44⫾7 apoptotic
nuclei/10 000 SM myocyte nuclei before to 49⫾7/ 10 000 at
6 months). The training-induced decrease in oxidative stress
(reduction in nitrotyrosine formation) was directly related to
the reduction in apoptosis (r⫽0.69, P⬍0.01; Figure 4).
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Figure 4. Association between change (6 months minus beginning) in nitrotyrosine formation as marker of oxidative stress and
change in number of apoptotic SM nuclei in vastus lateralis
muscle in CHF. Open circles represent CHF group C, and filled
circles, CHF group T. r⫽0.69 for patients in T (y⫽6.2x⫺6.1,
P⬍0.05), r⫽0.64 for patients in C (y⫽3.8x⫹1.9, P⬍0.05), r⫽0.8
for all CHF patients (y⫽6.4x⫺3.2, P⬍0.05).
Discussion
Two major findings emerge from this clinical study investigating the cross-talk between local inflammation and key
radical scavenger enzymes in the SM of patients with CHF:
(1) The increased expression of inflammatory cytokines is
associated with impaired activity of the radical scavenger
enzymes SOD, GPX, and Cat. The resulting amplification in
local oxidative stress contributes to enhanced muscle damage
by apoptosis. (2) Exercise training exerts unambiguous antiinflammatory and antioxidative effects in SM in CHF. The
training-induced reconstitution of GPX and Cat activity
culminates in decreased ROS-mediated protein modifications
and a reduction in SM apoptosis in CHF.
Expression of Radical Scavenger Enzymes in SM
of Patients With CHF
The enzyme SOD represents the first defense against superoxide anions and converts superoxide radicals to hydrogen
peroxide. In our study, we provide evidence that total SOD
activity is significantly reduced in SM of patients with CHF,
suggesting impaired detoxification of ROS. Unfortunately,
because of the small samples obtained from our patients, we
were unable to perform additional assays to distinguish the
activities of CuZnSOD, which is primarily found in the
cytosol, from those of MnSOD, which is mainly located in
the mitochondrial matrix, scavenging ROS produced by the
respiratory chain.17 Nevertheless, the reduction in total SOD
activity seems to be primarily the result of impaired MnSOD
expression, because CuZnSOD protein content was comparable between patients with CHF and HSs.
Hydrogen peroxides, end-products of the detoxification of
superoxide radical by SOD, are subjected to further decomposition by GPX.17 In the present study, GPX activity was
significantly attenuated by 51% in SM of CHF patients
compared with HSs; however, the reduction in GPX mRNA
content in concert with its decreased activity is consistent
with the hypothesis of a partial defect at the transcriptional
level in CHF. Furthermore, diminished tissue concentrations
of the necessary cofactor glutathione, seen in patients with
CHF, under the influence of elevated local levels of TNF-␣
could further aggravate the impaired GPX activity, leading to
ROS-mediated cell damage.11,20,21 This notion is strongly
supported by the inverse correlation between TNF-␣ protein
levels in SM and GPX activity in the present study. One
might argue that the increase in local TNF-␣ and IL-1␤
protein levels in CHF is the result of enhanced binding of
circulating cytokines on tissue-specific receptor. Indeed, serum concentrations of TNF-␣ were found to be elevated in
patient with CHF, whereas serum IL-1␤ levels remained
unaffected by CHF; however, the missing correlation between circulating and local levels of inflammatory cytokines,
in conjunction with the augmented mRNA levels of TNF-␣
and IL-1␤ in SM in CHF compared with HSs, strongly
supports our hypothesis of local inflammatory activation with
increased local expression of the aforementioned cytokines in
CHF.
The enzyme Cat primarily decomposes hydrogen peroxide
and shows, therefore, some overlap with the function of
GPX17; however, Cat converts hydrogen peroxide to water
and oxygen, independent of cellular glutathione concentrations and might, therefore, be specially important for the
elimination of hydrogen peroxide in CHF. In this study, the
remarkable decrease in Cat activity in SM of patients with
CHF was accompanied by a reduction in mRNA and protein
expression, suggesting a defect at the transcriptional level or
a reduction in Cat mRNA stability. Moreover, it is also
tempting to speculate that the decline in GPX activity results
in accumulation of hydrogen peroxide within the cell, finally
leading to substrate inhibition of Cat.29 The close inverse
correlation between TNF-␣ protein levels in SM and Cat
activity also suggests that this inflammatory cytokine inhibits
Cat directly, but the exact mechanism remains to be
determined.
In summary, these data are consistent with the hypothesis
that inflammatory cytokines that are locally expressed in SM
act as dual enhancers of oxidative stress: they induce the
expression of radical producers and inhibit the activity of
key radical scavengers.9,10,12 In the present study, elevation
of local oxidative stress was confirmed by the increase in
lipid peroxidation and nitrotyrosine formation in SM. The
accumulation of ROS has been shown to feed back on the
expression of cytokines and, hence, might contribute to
further acceleration of the inflammatory activation in
SM.1,9 –11,30 –32 The concept of ROS-mediated muscle fiber
damage in CHF is strongly supported by the correlation
between nitrotyrosine formation and the occurrence of
apoptotic cell death of SM myocytes in our study. Although we did not assess muscle function in the present
clinical trial, data from recent studies suggest that the
increases in local oxidative stress and apoptosis are associated with muscle dysfunction and loss of muscle bulk,
partially contributing to the typical clinical sign of early
muscle fatigue.14,15 Besides other derangements, the aforementioned alterations might at least partially contribute to
the development of cardiac cachexia in CHF.1,13–16,32,33
Linke et al
Influence of Exercise Training on Expression of
Radical Scavenger Enzymes in SM
Downloaded from http://circ.ahajournals.org/ by guest on January 13, 2017
Although Cat and GPX activities were significantly augmented after 6 months of exercise training in SM of CHF
patients, total SOD activity remained unchanged, suggesting
a persistent impairment of superoxide radical detoxification
in the SM. Because GPX mRNA expression was not changed
by exercise training in CHF, the increase in activity is the
result of either enhanced GPX mRNA stability or posttranslational modification of GPX protein. Nevertheless, we demonstrated that local TNF-␣ expression decreased in SM in
CHF in response to exercise training. Because TNF-␣ has
been shown to impair intracellular levels of the essential
cofactor of GPX (GSH), the attenuated local inflammation
after exercise training in CHF might be associated with
restoration of local tissue concentrations of GSH, thus contributing to partial restoration of GPX activity.20,21 This
hypothesis is supported by the close correlation between the
change in local TNF-␣ protein levels and the change in GPX
activity. Because GPX is able to convert hydrogen peroxide
to water and oxygen, it is tempting to speculate that the
training-induced augmentation of GPX activity results in a
reduction in cellular hydrogen peroxide levels and consequently, the elimination of substrate inhibition of Cat.29 This
might explain the increase in Cat and GPX activity in CHF
patients seen with exercise training, despite the fact that no
change occurred at the mRNA or protein level. Nevertheless,
because GPX and Cat expression was not normalized, these
data suggest that 6 months of exercise training are not
sufficient to correct the impaired transcription of GPX and
Cat in CHF.
To assess whether the decrease in TNF-␣ and IL-1␤
protein content in SM in CHF is secondary to alterations of
circulating cytokines, the latter were determined by ELISA.
Exercise training did not affect serum concentration of
TNF-␣ and IL-1␤ in patients with CHF. In contrast, we
observed a decrease in TNF-␣ and IL-1␤ mRNA expression
in SM in response to exercise training, which was associated
with a reduction in local TNF-␣ and IL-1␤ protein levels;
however, we did not find any correlation between circulating
TNF-␣ or IL-1␤ concentrations and the change in mRNA or
protein levels of the respective cytokines in SM in patients
with CHF undergoing exercise training. These finding are
consistent with the hypothesis of an established selfperpetuating vicious oxidative-inflammatory circle in SM in
CHF that is locally regulated and partially independent of the
circulating cytokine network.
The concept that exercise training in CHF exerts long-term
antioxidative effects because of correction of the enzymatic
radical scavenger system is further supported by the convincing normalization of lipid peroxidation and a reduction in
nitrotyrosine formation, as measures of local oxidative stress,
seen in SM of CHF patients after 6 months of exercise
training in the present study. This attenuation in local oxidative stress was closely associated with a decrease in the rate
of SM apoptosis in CHF to values normally determined in
healthy individuals. Nevertheless, there was no direct correlation between the change in Cat or GPX activity and the
change in local oxidative stress in the training group, which is
Radical Scavenger Enzyme Activity in CHF
1769
not surprising because of the multifactorial effects of exercise
training.
Recently, in an elegant study, Ennezat and coworkers34
demonstrated an increase in mRNA abundance of GPX and
CuZnSOD in SM of patients with CHF after 12 weeks of
exercise training. In that study, mRNA transcripts of GPX
and CuZnSOD were normalized to those of vWF, a protein
that is almost exclusively found in endothelial cells. Because
CuZnSOD and GPX are expressed not only in endothelial
cells but also in skeletal myocytes and that the majority of
RNA in muscle biopsy samples is derived from the largest
proportion of cells, which are clearly myocytes, we normalized mRNA expression of the radical scavenger enzymes to
18S rRNA that is known to be ubiquitously expressed in all
cell types. Because of different normalization procedures
(18S rRNA versus vWF), the results of our study and those of
Ennezat et al cannot be directly compared.
Previously, we were able to show that patients with CHF
benefit from an aerobic endurance exercise training with
respect to endothelial function, central hemodynamics, and
reversal of peripheral alterations, in the absence of obvious
harmful side effects.3,4,19 Because of those positive experiences, patients with CHF were subjected to such a training
program in the present study. It is conceivable that resistance
training or a combination of resistance and endurance training
has higher antiinflammatory and antioxidative properties;
however, further studies are necessary to address this issue.
Limitations
In the present study, we examined the cross-talk between
local inflammation and expression as well as the activity of
radical scavenger enzymes in crude SM homogenates, which
consist of SM myocytes and small vessels. Therefore, we
were unable to elucidate the exact source of radical scavenger
enzymes and inflammatory cytokines. Moreover, we cannot
rule out the possibility that circulating cytokines bind to their
specific receptor on the surface of myocytes, thereby partially
contributing to the TNF-␣ and IL-1␤ signal detected by
immunohistochemistry. The missing correlation between circulating and local levels of inflammatory cytokines, however,
strongly argues against this hypothesis. Given that most of the
cells are of muscular origin (Figure 1), it is highly probable
that the measured differences with respect to mRNA, protein
content, and activity of radical scavenger enzymes and
cytokines are due to alterations in SM myocytes in CHF.
We assessed lipid peroxidation in SM, which can be
considered a cumulative marker of radical damage; however,
lipid peroxides can also be catalyzed enzymatically, and they
do not necessarily reflect the present radical load at the time
when the SM biopsy is obtained. Therefore, further studies
with spin traps are necessary to exactly elucidate sources and
consequences of increased radical production in CHF.
Clinical Implications
In general, these data suggest that exercise training in CHF
represents an adjuvant therapy with remarkable antiinflammatory and antioxidative properties: it not only decreases the
expression of inflammatory cytokines, but it also enhances
the activity of radical scavenger enzymes, leading to a clear
1770
Circulation
April 12, 2005
reduction of oxidative stress and an attenuation of SM
damage induced by apoptosis.
16.
Acknowledgments
This study was supported by grant Ha 2165/3-2 from the Deutsche
Forschungsgemeinschaft, Bonn, Germany. We thank Silke Krabbes,
Angela Kricke, and Jeanine Böger for excellent technical assistance.
17.
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Antioxidative Effects of Exercise Training in Patients With Chronic Heart Failure:
Increase in Radical Scavenger Enzyme Activity in Skeletal Muscle
Axel Linke, Volker Adams, Paul Christian Schulze, Sandra Erbs, Stephan Gielen, Eduard Fiehn,
Sven Möbius-Winkler, Andreas Schubert, Gerhard Schuler and Rainer Hambrecht
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Circulation. 2005;111:1763-1770; originally published online April 4, 2005;
doi: 10.1161/01.CIR.0000165503.08661.E5
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2005 American Heart Association, Inc. All rights reserved.
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