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Cardiology Unit, Department of Medicine, University of Rochester Medical Center, Rochester, New York 14642
Submitted 7 January 2003 ; accepted in final form 16 April 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-carotene, ascorbic acid, and 
-tocopherol),
-tocopherol alone, or placebo for 8 wk. Compared with sham-operated
animals, CHF animals exhibited increased oxidative stress as evidenced by
decreased myocardial reduced-to-oxidized glutathione (GSH/GSSG) ratio (27
± 7 vs. 143 ± 24, P < 0.05), myocyte apoptosis (77
± 18 vs. 17 ± 4 apoptotic nuclei/10,000 cardiomyocytes,
P < 0.05), increased total and phosphorylated c-Jun
NH2-terminal protein kinase (p-JNK; 1.95 ± 0.14 vs. 1.04
± 0.04 arbitrary units, P < 0.05) and phosphorylated p38
kinase (p-p38), and decreased phosphorylated extracellular signal-regulated
kinase (p-ERK). Administration of antioxidant vitamins and -tocopherol
attenuated oxidative stress, myocyte apoptosis, and cardiac dysfunction, with
reversal of the changes of total JNK, p-JNK, and p-ERK in CHF. Furthermore,
because NE infusion produced changes of JNK, p-p38, and p-ERK similar to those
in CHF, we conclude that NE may play an important role in the production of
oxidative stress, MAPK activation, and myocyte apoptosis in CHF.
congestive heart failure; signal transduction; norepinephrine; oxidative stress; mitogen-activated protein kinase
-carotene, ascorbic acid, and
-tocopherol) attenuate the NE-induced myocyte apoptosis. Likewise, we
have found (38) that oxidative
stress is increased in pacing-induced congestive heart failure (CHF) and that
the decrease of cardiac noradrenergic nerve terminal profiles in CHF could be
prevented by treatment with antioxidant vitamins. However, it is not known
whether the reduced cardiac function in CHF is linked to myocyte apoptosis or
how oxidative stress produces myocyte apoptosis in CHF or NE infusion.
Oxidative stress may activate multiple cell signaling pathways
(25), including the
well-characterized mitogen-activated protein kinase (MAPK) pathways in the
heart (14). Two of the MARK
pathways, c-Jun NH2-terminal protein kinase (JNK) and p38 kinase
(p38), are proapoptotic, whereas extracellular signal-regulated kinase (ERK)
is antiapoptotic (1,
42). In this present study, we
proposed to measure myocyte apoptosis in pacing-induced cardiomyopathy and to
determine whether the MAPK pathways are involved in the oxidative
stress-induced myocyte apoptosis. We measured both the total and
phosphorylated amounts of MAPKs. We also examined whether myocyte apoptosis
was associated with cardiac dysfunction in CHF. Animals were also administered
antioxidant vitamins (-carotene, ascorbic acid, and -tocopherol)
to determine whether the improvement of cardiac dysfunction produced by the
vitamins is linked to reversal of the changes in MAPK proteins or myocyte
apoptosis. To further investigate whether the MAPK pathways are involved in
myocyte apoptosis produced by NE-induced oxidative stress, we measured cardiac
oxidative stress, myocyte apoptosis, MAPK activity, and cardiac function in
animals treated with NE infusion with and without the treatment of the vitamin
E analog trolox C or superoxide dismutase.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The studies were approved by the University of Rochester Committee Animal Resources and conformed to the guiding principles approved by the Council of the American Physiological Society and the National Institutes of Health guide on the humane care and use of laboratory animals. Two experimental animal models were employed.
Pacing-induced CHF. Adult healthy New Zealand White rabbits
(2.6–3.6 kg) were chosen for experimental heart failure using a modified
rapid cardiac pacing technique
(26). Briefly, subxyphoid
thoracotomy and pericardiotomy were performed under isoflurane gas anesthesia
for placement of two shielded pacing leads (TPW 50; Ethicon, Somerville, NJ)
onto the left ventricular (LV) apex and the left pectoral muscle,
respectively. One week later, animals were randomly assigned to receive either
pacing at a rate of 360 beats/min with a Medtronic Prevail model 8086
programmable pacemaker (Minneapolis, MN) (CHF animals) modified for rapid
cardiac pacing for animal use or no cardiac pacing (sham animals). CHF rabbits
were divided randomly into three groups, according to the vitamin regiments:
1) -carotene, 20 mg; ascorbic acid, 200 mg; and
-tocopherol, 200 mg; 2) -tocopherol alone, 200 mg; and
3) placebo. The doses were designed to infuse evenly over 8 wk using
subcutaneous pellets (Innovative Research of America, Sarasota, FL). These
doses have been shown to reduce oxidative stress in CHF in rabbits
(38). Sham-operated animals
received either placebo only or a mixture of three antioxidant vitamins as in
CHF animals. Animals were examined weekly for clinical evidence of heart
failure. After 8 wk, cardiac pacing was discontinued, and the animals were
prepared for resting hemodynamic and echocardiographic measurements and
arterial plasma NE (see Echocardiographic and Hemodynamic
Measurements). Animals were then killed with intravenous pentobarbital
sodium (100 mg/kg). The heart was removed, weighed, and rinsed in ice-cold
oxygenated normal saline. The ventricles were separated from the septum and
weighed. Transmural samples from the left ventricle were processed immediately
or stored in liquid nitrogen for later analysis.
NE infusion. The effects of NE and antioxidants on the MAPK proteins were studied in adult ferrets, using the LV muscle samples that were collected from a previously published study (29). In that study, each animal received two sets of subcutaneous sustained-release pellets (Innovative Research of America), with the doses calculated to deliver evenly over 4 wk. The first set of pellets was either NE (40 mg) or vehicle. The second set of pellets contained either vitamin E derivative 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox C; Sigma-Aldrich, St. Louis, MO) (100 mg), polyethylene-conjugated recombinant human superoxide dismutase (PEG-rhSOD; OXIS Health Products, Portland, OR) (20 mg, 3,100 ± 250 U/mg), or placebo pellet. Four weeks later, animals were killed for study. The effects of NE and antioxidants on resting hemodynamics, cardiac oxidative stress, cardiomyocyte apoptosis, and protein expressions of Bcl-2 and Bax have been reported previously (29, 33). Tissue samples from the following five experimental groups were chosen for the MAPK study: 1) a vehicle and a placebo pellet, 2) a vehicle and a trolox C pellet, 3) a NE pellet and a placebo pellet, 4) a NE pellet and a trolox C pellet, and 5) a NE pellet and a PEG-rhSOD pellet.
The person conducting the cardiomyocyte apoptosis and MAPK experiments was blinded to the drug assignment in the studies.
Echocardiographic and Hemodynamic Measurements
Maximal LV end-diastolic dimension (EDD) and end-systolic dimension (ESD) were measured, using a 5-MHz transducer on a Toshiba model SSH-60A sonographic system (Toshiba America Medical System, Tustin, CA). LV fractional shortening (FS) was calculated as FS = [(EDD – ESD) x 100]/EDD.
For the hemodynamic studies, animals were anesthetized with intramuscular ketamine (35 mg/kg) and midazolam (0.8 mg/kg). A 20-gauge fluid-filled catheter (Insyte; Deseret Medical, Becton Dickinson, Sandy, UT) was introduced into the left carotid artery and connected to a pressure transducer (model P23XL; Spectramed, Oxnard, CA) for measuring aortic pressure. A 2-Fr micromanometer-tipped catheter (Millar Instruments, Houston, TX) was advanced into the left ventricle via the right carotid artery for measuring the LV pressure and the first derivative of the LV pressure (dP/dt) using an electronic differentiator. Electrocardiograms, aortic pressure, and the LV dP/dt were recorded on a Brush model 480 recorder (Gould Instruments System Division, Cleveland, OH). Resting hemodynamic measurements were obtained in triplicate over a 20-min steady-state period at least 1 h after the catheterization. Averages of triplicates were used for statistical analysis.
Myocardial Glutathione Measurement
Fresh LV myocardium was homogenized in 3 volumes of 1% picric acid, and the supernatant was collected for measuring total glutathione using a glutathione reductase-coupled enzymatic assay (17) on a Lambda 3 UV/VIS spectrophotometer (Perkin Elmer, Norwalk, CT). Oxidized glutathione (GSSG) was measured by masking the reduced glutathione (GSH) with 2-vinyl pyridine in the enzymatic assay. The ratio GSH/GSSG was calculated as total tissue oxidative stress.
Detection of Apoptosis by MAb to Single-Stranded DNA
Frozen tissue sections were fixed in 85% methanol in phosphate-buffered saline (PBS). The sections were incubated with mouse anti-single-stranded DNA MAb (Chemicon International, Temecula, CA). The sections were then incubated with biotin-conjugated anti-mouse IgM (Vector Laboratory, Burlingame, CA) and avidin and biotinylated horseradish peroxidase macromolecular complex (Vector Laboratory) and were stained with 3-amino-9-ethylcarbazole (Vector Laboratory) and hematoxylin (Vector Laboratory). For the positive control, muscle sections were incubated with proteinase K (20 µg/ml) (16). The samples were examined under light microscopy. Four sections randomly picked from each of four pieces were analyzed per animal. Cardiomyocyte nuclei were determined by random counting of 10 fields per section. The number of apoptotic nuclei was calculated per 10,000 cardiomyocytes.
TUNEL Assay
Ventricular muscle paraffin sections were fixed in 4% methanol-free formaldehyde solution in PBS. The sections were stained using the Apoptosis Detection System (Promega, Madison, WI) per the manufacturer's instructions. Briefly, the sections were incubated with terminal deoxynucleotidyl transferase and fluorescein-labeled dUTP. To identify cardiomyocytes, we incubated sections with mouse anti-myosin heavy chain MAb (Chemicon International). Finally, to identify all nuclei (nonapoptotic and apoptotic), sections were stained with propidium iodide (Sigma-Aldrich). The samples were analyzed under a fluorescence microscope. The number of apoptotic nuclei was determined as described in Detection of Apoptosis by MAb to Single-Stranded DNA.
Western Blot for MAPK Proteins
Ventricular muscle samples were homogenized and extracted for proteins. Aliquots containing 30–50 µg of protein were separated by electrophoresis through 10–12% SDS-polyacrylamide gel. Equal loading of protein was confirmed by Coomassie blue staining. Mouse anti-JNK, anti-ERK, and anti-p38 MAbs (Santa Cruz Biotechnology, Santa Cruz, CA) were used to detect total protein levels of JNK, ERK, and p38, respectively. The Phototope-HRP Western blot detection kit (New England Biolab, Beverly, MA) was used to visualize the bands. The Western blot signals were scanned by GS-700 imaging densitometer, and the bands were quantified using the Quantity One program (Bio-Rad Laboratories, Hercules, CA). The optical density of tissue samples was normalized to a control sample in arbitrary densitometry unit.
MAPK Protein Kinase Activity Assay
The functional activity of JNK, ERK, and p38 kinases was studied using phospho-specific antibodies (p-JNK, p-ERK, and p-p38) (Santa Cruz Biotechnology). Protein extracts (40–60 µg) were resolved by 12% SDS-polyacrylamide gel and transferred to polyvinylidene fluoride membranes. The membranes were then probed with p-JNK, p-ERK, and p-p38 and analyzed as described in Western Blot for MAPK Proteins.
Statistical Analysis
Results are presented as means ± SE. The statistical significance of differences among experimental groups or between two means was determined using analysis of variance and multiple comparison tests (RS/1 Research System; Bolt, Beranek and Newman Software Products, Cambridge, MA). Pearson product-moment correlation analysis was used to determine the relationship between cardiomyocyte apoptosis and cardiac function (LV dP/dt and LV FS). P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Cardiac function and resting hemodynamics. The effects of rapid
ventricular pacing and antioxidant vitamins on body and heart weights, resting
hemodynamics, and plasma NE are shown in
Table 1. Rapid ventricular
pacing produced no significant changes in body or heart weight. Cardiac
dysfunction was evidenced in these animals by increased right atrial pressure
and LV end-diastolic pressure and dimension and decreased mean aortic blood
pressure, LV dP/dt, and LV FS. Administration of antioxidant vitamins
had no effects on body weight, heart rate, mean aortic blood pressure, right
atrial pressure, and LV end-diastolic pressure in sham or CHF animals but
attenuated the increase of LV end-diastolic pressure and decreases of LV FS
and LV dP/dt in CHF. Similar changes occurred in LV end-diastolic
pressure, FS, and dP/dt of CHF animals after -tocopherol
treatment alone. However, the magnitude of effects of -tocopherol on LV
end-diastolic pressure and FS was smaller than that produced by vitamin
combination. Plasma NE was significantly elevated in CHF animals. Antioxidant
vitamins did not affect the increase of NE concentration in either
sham-operated or CHF animals.
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Myocardial glutathione. Rapid ventricular pacing increased
myocardial GSSG content and decreased the GSH/GSSG ratio
(Fig. 1). Antioxidant vitamins,
which had no effects in sham animals, attenuated the changes in myocardial
GSH/GSSG ratio in CHF. -Tocopherol also reduced the decrease of
myocardial GSH/GSSG ratio in CHF animals, but the effect was smaller than that
produced by vitamin mixtures.
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Myocyte apoptosis by MAb to single-stranded DNA.
Figure 2A shows the
positive control of immunohistochemical staining of single-stranded DNA of
rabbit ventricular muscle section after proteinase K treatment. Representative
pictures of the MAb staining of ventricular sections from a sham, CHF, and CHF
plus vitamins animal are shown in Fig. 2,
B–D. Figure
2E shows the group data. The number of MAb-positive
nuclei was increased in CHF compared with the sham animals. Administration of
antioxidant vitamins or -tocopherol had no effect in sham rabbits but
reduced the number of apoptotic myocyte nuclei in CHF animals.
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Total JNK, ERK, and p38 by Western blot. Total JNK was increased
in CHF (1.8 ± 0.2 arbitrary units) compared with sham animals (1.0
± 0.1 arbitrary units, P < 0.001). This increase of total
JNK in CHF was abolished by administration of antioxidant vitamins (1.1
± 0.1 arbitrary units) or -tocopherol (1.2 ± 0.1
arbitrary units). In contrast, total ERK and p38 did not differ between the
sham and CHF animals, and antioxidant vitamins or -tocopherol alone did
not affect the total protein expression of ERK or p38.
Activation of phosphorylation of JNK, ERK, and p38. The
phosphorylated active forms of JNK were detected in rabbit heart as bands
corresponding to 46 kDa (p-JNK1) and 54 kDa (p-JNK2). The activity of p-JNK1/2
was increased in CHF (Figure
3A). This increase was prevented by antioxidant vitamins
or -tocopherol in CHF (Fig. 3,
A and B). ERK has two phosphorylated active
forms detected as a 44-kDa band (p-ERK1) and a 42-kDa band (p-ERK2)
(Fig. 3C). Rapid
ventricular pacing reduced p-ERK1/2 activity. Antioxidant vitamins or
-tocopherol prevented the reduction of p-ERK1/2 in CHF
(Fig. 3, C and
D) but had no effects in sham animals. Like p-JNK1/2,
p-p38 was increased in CHF (Fig. 3,
E and F). However, unlike p-JNK1/2, p-p38
increased further after administration of antioxidant vitamins or
-tocopherol in CHF and sham animals.
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Correlation between cardiac apoptosis and function in CHF. Significant negative correlations were observed between cardiomyocyte apoptosis and LV dP/dt (Fig. 4A) and between cardiomyocyte apoptosis and LV FS (Fig. 4B). The quantities r2 indicate that 33–49% of the variances of LV dP/dt and LV FS could have been accounted for by the variation of myocyte apoptosis.
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Chronic NE Administration
We reported previously (29) that there were no significant differences in body weight, heart weight, heart rate, mean aortic pressure, or LV dP/dt in vehicle- and NE-treated ferrets with and without trolox C or PEG-rhSOD. However, plasma NE increased fourfold in ferrets receiving subhypertensive doses of NE (29, 33). This resulted in an increase in oxidative stress in the heart, as evidenced by increased myocardial GSSG and decreased GSH/GSSG ratio. Administration of trolox C or PEG-rhSOD abolished the decrease of GSH/GSSG in NE-treated ferrets but had no effects on plasma NE concentration in either vehicle- or NE-treated animals. We did not find any significant differences in body weight, LV weight, heart rate, mean aortic pressure, and LV dP/dt between vehicle- and NE-treated ferrets with and without trolox C or rhSOD.
Figure 5 shows the
TdT-mediated dUTP nick-end labeling (TUNEL) labeling in ferret myocardium.
Apoptotic myocytes were identified by double labeling, showing colocalization
of the fragmented DNA and -heavy chain myosin within the same cells.
Figure 6 shows the increases of
MAb-positive and TUNEL-positive nuclei in NE-treated animals compared with
vehicle-treated animals. Administration of trolox C or rhSOD, which had no
effect in vehicle-treated animals, reduced the numbers of MAb-positive and
TUNEL-positive nuclei in the NE-treated animals.
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NE administration increased total JNK protein (1.4 ± 1 arbitrary units) compared with the vehicle control (1.0 ± 0.1 arbitrary units, P < 0.01) but had no effect on either total p38 or ERK. The increase in total JNK seen in NE-treated animal was abolished by trolox C (1.0 ± 1 arbitrary units) or rhSOD (1.1 ± 0.1 arbitrary units). Neither trolox C nor rhSOD affected total p38 or ERK protein in vehicle- or NE-treated animals (data not shown).
The effects of NE on myocardial phospho-MAPKs are shown in Fig. 7. Similar to the changes in total JNK, NE administration increased p-JNK1/2, and this increase was abolished by addition of either trolox C or rhSOD (Fig. 7, A and B). NE administration decreased p-ERK1/2, and the decrease was attenuated by trolox C or rhSOD (Fig. 7, C and D). Neither trolox C nor rhSOD had an effect on p-JNK1/2 or p-ERK1/2 in vehicle-treated animals. Finally, p-p38 was increased in the NE-treated animals (Fig. 7, E and F). Trolox C or rhSOD treatment produced a further increase in p-p38 in both NE- and vehicle-treated animals.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-tocopherol attenuated cardiac oxidative stress and myocyte apoptosis
in CHF, and these effects were associated with reversal of the changes of JNK
and ERK activity. Similarly, cardiomyocyte apoptosis produced by NE infusion
was associated with increased cardiac oxidative stress, increased JNK
activity, and decreased ERK activity. Like antioxidant vitamins in CHF,
administration of trolox C or superoxide dismutase inhibited oxidative stress
and myocyte apoptosis and attenuated the elevation of JNK activity and the
decline of ERK activity in ferrets treated with NE. The findings suggest that
the increased cardiac oxidative stress in CHF is mediated, at least in part,
via the increased NE in the heart and that myocyte apoptosis in CHF probably
is caused by activation of JNK and inactivation of ERK. The findings further
suggest that antioxidant therapy may be beneficial in CHF by reducing
oxidative stress and myocyte apoptosis via the JNK and ERK pathways. Oxidative Stress and Apoptosis in CHF
Myocyte apoptosis occurs in experimental and human heart failure
(24,
32). Our present study
confirms that myocyte apoptosis occurs in CHF produced by rapid pacing. This
action is mediated, at least in part, by oxidative stress known to occur in
CHF (4,
40).
Figure 8 illustrates a
simplified version of the events, linking heart failure to oxidative stress
and cardiomyocyte apoptosis. Earlier studies have shown that hydroxyl free
radical formation is increased in failing heart
(20–22)
and that the increase of reactive oxygen species is capable of inducing
apoptosis in cultured myocytes
(12) and cardiac dysfunction
in intact heart (20,
27). Rapid ventricular pacing
also has been shown to increase oxidized products of mitochondrial DNA
(38) and mitochondrial damage
(22). Reactive oxygen products
such as nitrotyrosine and potentiation of the oxidative stress response by p66
(Shc) also have been shown to enhance myocyte apoptosis in pacing-induced
heart failure (7). The
importance of free radical formation in myocyte apoptosis is further supported
by the findings that a number of antioxidants can reduce or block myocyte
apoptosis in heart failure
(25,
27). The oxygen free radicals
may be derived from mitochondrial electron transport chain, NADH/NADPH
oxidase, xanthine oxidase, nitric oxide synthase, and/or cyclooxygenase
(39). Other factors such as
catecholamines, angiotensin II, and tumor necrosis factor- also have
been implicated in myocyte apoptosis in heart failure
(19).
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Signaling Pathways of Oxidative Stress-Induced Myocyte Apoptosis in CHF
Prior studies have shown that MAPKs are stimulated by oxidative stress in cultured myocytes (10) and isolated perfused hearts (9). MAPKs are also activated during transition from hypertrophy to heart failure (18). Our present study shows that the increased apoptosis in the pacing-induced cardiomyopathy is associated with an increase in JNK activity and a decrease in ERK activity (Fig. 8). These findings are consistent with prior observations in myocardial ischemia and reperfusion (28, 43). As reported in this study, antioxidants reduce the activity of MAPKs in myocardial ischemia-reperfusion, further supporting a functional role of MAPK in the oxidative stress-induced myocardial damage (36). Furthermore, JNK activation has been shown to mediate oxidative stress-induced caspase activation (42). The results are further supported by the recent studies in cultured cardiomyocytes showing that oxidative stress-induced JNK activation causes the release of cytochrome c from mitochondria to cytosol, leading to caspase 9 activation and apoptosis (3) and that superoxide dismutase mimetic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride reduces mitochondrial cytochrome c release and apoptosis mediated via inhibition of JNK (34). These findings suggest that oxidative stress-mediated JNK activation is associated with mitochondrial cytochrome c release, caspase activation, and apoptosis (Fig. 8).
Early studies have shown that p38 is proapoptotic
(30), but in a recent study
(15), functional recovery of
the failing heart by mechanical unloading was associated with reduced myocyte
apoptosis and increased p38 activity. The difference may result from the
selective activation of p38 isoforms. p38 activation causes apoptosis,
whereas p38 activation induces hypertrophy and survival
(37). In our present study,
cardiac p-p38 was increased by antioxidant vitamins and superoxide dismutase,
which reduced cardiomyocyte apoptosis in CHF, suggesting that the
cytoprotection probably is conferred by p38 activation in CHF.
Unlike the changes that could be produced by large doses of H2O2 in cultured myocytes (1, 10), the activity of MAPKs changed modestly (20–50%) in intact animals as shown in our present study of CHF and chronic NE administration. However, these modest changes of MAPK activity in intact animals probably are physiologically significant in myocyte apoptosis and LV function in our study. Similar changes of antiapoptotic Bcl-2 and proapoptotic Bax proteins also have been shown to contribute to myocyte apoptosis in NE infusion (33) and after myocardial ischemia and reperfusion (45).
Reduction of Apoptosis by Antioxidants is Associated With Improvement of Cardiac Function in CHF
Myocyte apoptosis plays a role in the transition from LV hypertrophy to heart failure after chronic pressure overload (11). There is also a correlation between the severity of heart failure and myocyte susceptibility to apoptosis in patients with dilated cardiomyopathy (2) and rats with adriamycin-induced cardiomyopathy (31). In the present study, although antioxidant vitamins abolished the changes in JNK and ERK and apoptosis in CHF, LV mechanical function was only partly reversed, indicating that other factors were operative in cardiac dysfunction in pacing-induced heart failure. The persistent rapid ventricular pacing could have masked the functional improvement of cardiac muscle function. Our findings indicate a functional linkage between oxidative stress-induced MAPK changes and apoptosis and cardiac dysfunction in CHF and support the concept that oxidative stress-mediated MAPK changes and myocyte apoptosis contribute to cardiac dysfunction. The findings are consistent with the observation that overexpression of antiapoptotic Bcl-2 reduces myocyte apoptosis and improves cardiac function after myocardial ischemia-reperfusion (8). The studies indicate that antioxidant therapy that interferes with the induction of myocyte apoptosis may provide a novel therapeutic target in CHF.
However, the use of antioxidants (e.g., vitamin E) in cardiovascular diseases remains controversial. An inverse association has been observed between antioxidant intake or body status and the risk of cardiovascular diseases (6, 13). The recently reported Antioxidant Supplementation in Atherosclerosis Prevention Study (35) confirms that the combination of vitamin E and slow-release vitamin C slows the progression of atherosclerosis in hypercholesterolemic subjects. In the Cambridge Heart Antioxidant Study (41), vitamin E treatment reduced the risk of nonfatal myocardial infarction in patients, but there was no significant effect on cardiovascular mortality. In the Heart Outcome Prevention Evaluation Study (44), vitamin E supplementation failed to show beneficial cardiovascular effects in high-risk subjects. The reasons for the discrepant results are not known but may relate to the different patient characteristics and the doses of antioxidants (23). The effects of antioxidant vitamins on human CHF require further clarification.
The doses of antioxidant vitamins used in the present study were based on
our previous studies (29,
38). The doses are 3–10
times the current recommended dietary allowances for humans and are within the
human therapeutic ranges based on body weight
(5). They were sufficient to
increase myocardial -tocopherol by 70% and decrease oxidative stress as
judged by the changes in tissue GSH/GSSG ratio
(38). Beneficial effects of
the antioxidant vitamins at the present doses are also evidenced by the
improvements of cardiac sympathetic nerve terminal function and
-adrenergic receptor downregulation in NE infusion
(29) and CHF animals
(38). The findings suggest
that the doses of vitamins we chose for the study are both clinically relevant
and pharmacologically sufficient to produce desired antioxidant effects.
However, oxidative stress may induce leakage of mitochondrial cytochrome
c, activation of the caspase 3, and myocyte apoptosis, independently
of the MAPK pathway (Fig. 8).
These possibilities require further investigations. Further studies are also
needed to establish the molecular mechanisms by which the antioxidant vitamins
exert the beneficial effects on the MAPK signal pathways and myocyte
apoptosis.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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This work was presented in part before at the 2001 Annual Scientific Sessions of the American Heart Association, Anaheim, CA, on November 14, 2001.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.05 vs. CHF placebo group.
P < 0.05 vs. sham vitamins group.
