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Am J Physiol Heart Circ Physiol 284: H442–H448, 2003;
10.1152/ajpheart.00560.2002.
Reduced inotropic heart response in selenium-deficient mice
relates with inducible nitric oxide synthase
RICARDO M. GOMEZ,1 ORVILLE A. LEVANDER,2 AND LEONOR STERIN-BORDA1
Cátedra de Farmacologı́a, Facultad de Odontologı́a, Universidad de Buenos Aires,
Consejo Nacional de Investigaciones Cientı́ficas y Técnicas de la República Argentina,
1122 Buenos Aires, Argentina; and 2Beltsville Human Nutrition Research Center,
Department of Agriculture/Agricultural Research Center, Beltsville, Maryland 20705
1
Submitted 4 October 2002; accepted in final form 5 October 2002
antioxidants; cardiomyopathy; isoproterenol; Keshan disease
SELENIUM (Se) is an essential micronutrient with antioxidant functions because it is incorporated as selenocysteine into proteins that protect against oxidative
stress (44). Low dietary Se intake has been associated
with pathologies frequently involving muscle in animals of agricultural value as well as in some human
diseases (11). Of particular interest is the link established between Se deficiency and an endemic human
dilated cardiomyopathy (DCM) known as Keshan disease, which is found in areas of China with Se-poor
soils (17). Although Se deficiency accounted for most
features of the disease, some infectious agents were
later suggested as etiological cofactors (43). Other epidemiological studies in Europe also have linked low
serum Se levels to cardiac disease (37, 38) and Se
supplementation has a protective effect during myocardial ischemia (33). The causal mechanisms by which Se
deficiency contributes to heart disease are not clearly
Address for reprint requests and other correspondence: L. SterinBorda, Pharmacology Unit, School of Dentistry, Univ. of Buenos
Aires, Marcelo T. de Alvear 2142, 4 “B” 1122AAH Buenos Aires,
Argentina (E-mail: [email protected]).
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understood, although it is generally agreed that increased free radical damage may play an important
role (9, 31).
Nitric oxide (NO) is a short-lived molecular free
radical synthesized from L-arginine by the catalytic
reaction of NO synthases (NOS). The mammalian NOS
isoforms include two constitutively expressed enzymes,
the neuronal NOS and the endothelial NOS (eNOS), as
well as the inducible isoform (iNOS). Both constitutive
NOS (cNOS) isoforms are regulated predominantly at
the posttranslational level, whereas iNOS appears to
be regulated primarily by the rate of transcription (5,
42). NO produced by either cNOS or iNOS influences
normal cardiac function and may play an important
role in the pathophysiology of certain disease states
associated with cardiac dysfunction (20).
In this study, we demonstrate that atria taken from
mice fed a Se-deficient diet (Se⫺) have a diminished
isoproterenol (Iso) or norepinephrine (NE)-induced
␤-adrenoceptor inotropic cardiac response compared
with atria from Se-adequate (Se⫹) mice. This diminished response could be reversed either by feeding the
Se⫺ mice the Se⫹ diet for 1 wk or by prior treatment
with NOS inhibitors. To our knowledge, this is the first
report that dietary Se deficiency causes an NO-related
depression of mouse atrial contractility stimulated by
an adrenergic agent. These results could have important implications for the possible role of Se in preventing certain heart diseases.
METHODS
Animals. Three-week-old outbred F1 mice of either gender
were randomly divided in two groups and fed either a Se⫺ or
a Se-supplemented diet for 4 wk before the experiment. They
were housed in stainless steel, wire-bottomed cages at a
density of 3–5 per cage and maintained at an ambient air
temperature of 22 ⫾ 1°C and a 12:12-h light-dark cycle. All
mice were given water and diets ad libitum. All procedures
used in the experiments were approved by the Ethics Committee of the Faculty of Odontology, University of Buenos
Aires.
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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Gomez, Ricardo M., Orville A. Levander, and Leonor
Sterin-Borda. Reduced inotropic heart response in seleniumdeficient mice relates with inducible nitric oxide synthase. Am J
Physiol Heart Circ Physiol 284: H442–H448, 2003; 10.1152/
ajpheart.00560.2002.—Atria from mice fed a selenium-deficient
(Se⫺) diet have a diminished ␤-adrenoceptor-inotropic cardiac
response to isoproterenol or norepinephrine compared with
atria from mice fed the same diet supplemented with 0.2 mg/kg
Se as sodium selenite (Se⫹). This diminished response could be
reversed by feeding Se⫺ mice the Se⫹ diet for 1 wk or by
pretreatment with nitric oxide synthase (NOS) inhibitors such
as NG-monomethyl-L-arginine or aminopyridine. Elevated serum concentrations of nitrite/nitrate as well as a threefold
increase in the atrial NOS activity were seen in the Se⫺ versus
Se⫹ mice. Western blotting and indirect immunofluorescence
indicated an enhanced expression of inducible NOS in hearts
from Se⫺ mice. Increased expression and activity of NOS and
increased nitrite/nitrate levels from Se⫺ mice correlated with
an impaired response to ␤-adrenoceptor inotropic cardiac stimulation. Elevated nitric oxide levels may account for some of the
pathophysiological effects of Se deficiency on the heart.
SELENIUM, NITRIC OXIDE, AND HEART CONTRACTILITY
AJP-Heart Circ Physiol • VOL
anti-iNOS antibody (Cayman), a biotinylated anti-rabbit antiserum (Vector), and a streptavidin peroxidase (Vector),
intercalated with several washes. Blots were developed using
50 ml of PBS with 20 mg of 3,3⬘-diaminobenzidine tetrahydrochloride (Sigma) and 15 ␮l of hydrogen peroxide (30%).
Nitrite/nitrate analysis. Serum samples from Se⫹ and Se⫺
mice were collected and stored at ⫺70°C until analysis. After
sample deproteinization, nitrite/nitrate levels were measured with a commercial kit (Calbiochem). Briefly, nitrate
was converted to nitrite by incubation with nitrate reductase
in the presence of NADPH. Lactate dehydrogenase was then
used to destroy excess NADPH. Equal volumes of sample and
Griess reagent were incubated at room temperature. After 10
min, absorbance was read at 550 nm. The nitrite concentration was determined by using sodium nitrate as a standard.
Indirect immunofluorescence. Heart samples from Se⫺ and
Se⫹ mice were quick frozen in isopentane that had been
chilled in liquid nitrogen, and 8-␮m cryostat sections were
prepared, air dried for 10 min, and stored at ⫺70°C until use.
To reduce background, slides were incubated for 20 min with
a blocking solution (1 M PBS with 10% goat serum and 0.5%
casein). A 1:100 dilution of a rabbit anti-iNOS antibody
(Cayman) was applied to the sections for 2 h at room temperature, washed for 15 min with PBS, and then incubated
for 30 min with a FITC-conjugated goat anti-rabbit IgG
(Vector). After several washes, the sections were photographed with a Nikon photomicroscope equipped with epiluminescence. As a negative control of the reaction, the first
antiserum was omitted.
Drugs. Iso, NE, AP, and L-NMMA were purchased from
Sigma. Stock solutions were freshly prepared in the corresponding buffers. The drugs were diluted in the bath to
achieve the final concentration stated in the text.
Statistical analyses. The data in the dose-response curves
(Figs. 1 and 2) were analyzed with the use of a nonlinear
mixed ANOVA logistic regression model (Proc NLN,
SAS Institute; Cary, NC). The data in Fig. 3, A and B, were
analyzed by a two-way and one-way ANOVA, respectively. Differences were considered statistically significant
if P ⬍ 0.05.
Fig. 1. Decreased contractility in force over time (dF/dt) of atria from
selenium-deficient (Se⫺) mice (F) in the presence of increasing concentrations of isopoterenol (Iso) (A) or norepinephrine (NE) (B) compared with the response of selenium-adequate (Se⫹) mice (■) or Se⫺
mice fed the Se⫹ diet (Se⫹/⫺) for 1 wk before the experiment (E). The
tissues were equilibrated for 30 min before the addition of agonist.
Values are expressed as the percent change compared with the basal
value before the addition of agonist and represent means ⫾ SE (n ⫽
6). * Parameters composing the logistic regression model for the Se⫺
diet group were significantly different from those composing the
models for the Se⫹ or Se⫺/⫹ groups.
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Diets. The composition of the diets has been previously
described (16). Briefly, the basal Se⫺ diet consisted of (in
percent) 30 torula yeast, 4 tocopherol-stripped lard, 1 tocopherol-stripped corn oil, 3.5 AIN-76 salt mix without Se, 1
AIN-76A vitamin mix without vitamin E, 0.3 DL-methionine,
0.152 choline dihydrogen citrate, and 60 sucrose. Vitamin E
(35 mg/kg) was added as all natural-␣-tocopherol acetate. For
Se⫹ diets, Se was added to the basal diet at 0.2 mg/kg as
sodium selenite. Before experiments, sera from mice were
analyzed for Se concentration to confirm Se status, as previously described (29).
Atrial preparation for contractility. The mice were euthanized by cervical dislocation. The atria were carefully dissected from the ventricles, attached to a glass holder, and
immersed in a tissue bath containing Krebs-Ringer bicarbonate (KRB) solution gassed with 5% CO2 in O2 and maintained
at pH 7.4 and 30°C. KRB solution was composed as described
previously (40). A preload tension of 350 g was applied to the
atria, and tissues were allowed to equilibrate for 30 min. The
initial control values for contractile tension of the isolated
atria were recorded using a force transducer coupled to an
ink-writing oscillograph (6). The preparations were paced
with a bipolar electrode and a stimulator (model SK4, Grass).
The stimuli had a duration of 2 ms and the voltage was 10%
above threshold. Inotropic effects were assessed by recording
the maximum rate of isometric force development over time
(dF/dt) during electrical stimulation at a fixed frequency of
300 beats/min. Control values (⫽100%) refer to the dF/dt
before the addition of drugs. The absolute value for dF/dt at
the end of the equilibration period (30 min) was 4.3 ⫾ 1.2 g/s.
Cumulative dose-response curves of Iso or NE were done on
atria from Se⫺ and Se⫹ mice according to a method previously described (46). A maximal effect was achieved within 5
min after each dose. When blockers were used, the atria were
incubated previously for 30 min before dose-response curves
of the agonists were done. In contrast, mice administered
2-amino-4-methylpyridine (AP) in vivo were injected subcutaneously at a daily dose of 0.3 mg 䡠 kg⫺1 䡠 day⫺1 for 3 days
before they were euthanized. Controls were mice injected
with PBS with the use of the same protocol.
Determination of NOS activity. NOS activity was measured in atria by production of [U-14C]citrulline from
[U-14C]arginine according to the procedure described originally for brain slices (8) modified for isolated rat atria (41).
Briefly, after 20 min of preincubation in KRB solution, the
atria were transferred to 500 ␮l of prewarmed KRB equilibrated with 5% CO2 in O2 in the presence of [U-14C]arginine
(0.5 ␮Ci). Appropriate concentrations of drugs were added
and the atria were incubated for an additional 20 min in the
same buffer. Atria were then homogenized with the use of an
UltraTurrax tissue dispersion system, and, after centrifugation at 20,000 g for 10 min at 4°C, supernatants were applied
to 2-ml columns of Dowex AG 50 WX-8 (sodium form);
[14C]citrulline was eluted with 3 ml of water and quantified
by liquid scintillation counting. When partial purification of
NOS was required, it was done as previously described (41).
Measurement of basal NOS activity in whole atria by the
above mentioned procedure was inhibited 95% in the presence of 5 ⫻ 10⫺4 M of NG-monomethyl-L-arginine (L-NMMA).
Western blot. Heart tissues from Se⫺ and Se⫹ mice were
homogenized in PBS with an UltraTurrax, aliquoted, and
frozen at ⫺70°C until use. Samples were boiled for 5 min and
electrophoresed on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Millipore HATF 20200)
at 4°C. Equal loading and transfer of proteins between lanes
was verified by Ponceau red staining before immunodetection. Blots were then probed consecutively with a rabbit
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SELENIUM, NITRIC OXIDE, AND HEART CONTRACTILITY
RESULTS
Fig. 2. Effect of NG-monomethyl-L-arginine (L-NMMA) (A) or 2amino-4-methylpyridine (AP) (B) on the dose-response curve of Iso on
atrial dF/dt. Tissues were incubated for 30 min in the absence (Se⫺
F; Se⫹ ■) or presence (Se⫺ E; Se⫹ 䊐) of the nitric oxide synthase (NOS)
inhibitor and then the dose-response curves to Iso were determined.
Values represent the means ⫾ SE (n ⫽ 6). * Parameters composing
the logistic regression model for the Se⫺ diet group in the absence of
inhibitors were significantly different from those composing the
models for the other groups.
AJP-Heart Circ Physiol • VOL
Fig. 3. A: atrial NOS activity in the absence of NOS inhibitors (solid
bars), in AP-treated mice (open bars), or in the presence of L-NMMA
(gray bars) in atria from Se⫹ mice, Se⫺ mice, or Se⫹/⫺ mice. aThe
differences between the Se⫺ atria without inhibitors and the other
values were significant (two-way ANOVA). B: nitrite/nitrate concentrations of sera from Se⫹, Se⫺, and Se⫺/⫹ mice. Values represent
means ⫾ SE (n ⫽ 6). bValue of Se⫺ group significantly greater than
those of the other 2 groups (one-way ANOVA).
These results encouraged us to determine whether
higher NO levels were involved in the diminished response of Se⫺ atria to ␤-agonists. In this regard, we
observed a higher NOS activity in the atria of Se⫺ mice
when compared with atria of Se⫹ mice (Fig. 3A). The
Se⫺ mice, which had been switched to a Se⫹ diet for 1
wk before the experiment (Se⫹/⫺), showed a NOS activity level similar to that of the Se⫹ mice that had
been fed the Se⫹ diet throughout the 4-wk feeding
period, thereby demonstrating the relationship between the activity of NOS and the type of diet. Administration of AP decreased atrial NOS activity from Se⫺
mice to levels seen in atria from Se⫹ and Se⫹/⫺ mice,
which remained unchanged by AP treatment. In contrast, L-NMMA (5 ⫻ 10⫺4 M) inhibited 95% of the NOS
activity in all three mouse groups (Fig. 3A). It is important to emphasize that in contractility experiments,
⫺6
L-NMMA was used at 5 ⫻ 10
M, a concentration
known to inhibit basal NOS activity by 50% without
modifying basal dF/dt (41). Significantly higher concentrations of nitrite/nitrate were found in the serum
from Se⫺ mice when compared with those from Se⫹
mice (Fig. 3B). Again, Se⫺ mice, which had been fed a
Se⫹ diet for 1 wk previously to the serum nitrite/
nitrate determination (Se⫹/⫺ mice), showed lower levels similar to those from the Se⫹ mice.
To establish whether the elevated levels of NO
associated with the diminished response to ␤-agonists observed in atria from Se⫺ mice could be generated by the iNOS, we looked for expression of the
enzyme by both indirect immunofluorescence and
Western blot procedures. We observed a low background fluorescence signal in Se⫹ hearts when tested
for iNOS expression (Fig. 4A). The staining is clearly
enhanced in Se⫺ hearts but retained its apparent
cytoplasmic localization (Fig. 4B). Expression of
iNOS in heart tissue samples was confirmed by
Western blot (Fig. 5).
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Serum Se concentrations were significantly (P ⬍
0.05) lower in mice fed the Se⫺ diet versus those fed the
Se⫹ diet (27 ⫾ 14 vs. 411 ⫾ 37 ng/ml, respectively; n ⫽
5), but there were no significant differences in body
weight between treatment groups (data not shown).
To determine whether dietary Se had any influence
on the mechanical response to ␤-agonists, the effects of
Iso and NE on dF/dt of atria taken from Se⫹ and Se⫺
mice were examined. The basal absolute dF/dt values
(g/s) of atria from Se⫺ and Se⫹ mice (before the addition of the ␤-agonists) were 4.3 ⫾ 1.2 for Se⫹ and 4.0 ⫾
1.1 for Se⫺ (n ⫽ 6). Figure 1 shows the effect of
increasing concentrations of Iso (Fig. 1A) and NE (Fig.
1B) on the contractility of atria from Se⫺ and Se⫹ mice.
Both Iso and NE induced a concentration-dependent
increase in atrial dF/dt, but in Se⫺ atria the agonistic
dose-response curve was shifted to the right and the
potency of the agonist was decreased. When Se⫺ mice
were fed the Se⫹ diet for 1 wk before the contractility
assay, the diminished dF/dt responses were abolished
and reached values similar to those of the Se⫹ mice
(Fig. 1, A and B). This observation strengthened the
causal relationship between the diminished dF/dt response of isolated atria to ␤-agonists and the Se⫺ diet.
To determine whether endogenous NO participated
in the decreased response of Se⫺ atria to ␤-agonists,
tissue was treated with different NOS inhibitors. The
inhibition of all NOS isoenzymes with L-NMMA (5 ⫻
10⫺6 M) increased the efficacy of Iso and shifted its
dose-response curve to the left so that the dF/dt values
of Se⫺ atria were similar to those obtained with Se⫹
atria, treated with L-NMMA or not (Fig. 2A). Similarly,
in vivo treatment of Se⫺ atria with AP, an inhibitor of
the iNOS isoenzyme, improved the positive inotropic
effect of Iso so that dF/dt values similar to those seen
with Se⫹ atria were obtained (Fig. 2B). At the concentrations used, all inhibitors had no effect per se on
basal contractility.
SELENIUM, NITRIC OXIDE, AND HEART CONTRACTILITY
Fig. 4. A: hearts from Se⫹ mice exhibit low indirect immunofluorescence signal due to inducible NOS (iNOS) in cardiomyocytes. B:
staining due to iNOS is clearly enhanced in hearts of Se⫺ mice with
a cellular localization similar to that of Se⫹ mice (magnification
⫻400).
DISCUSSION
AJP-Heart Circ Physiol • VOL
effect of NO on agonist-stimulated cardiac contractility
could be a direct interaction between NO and catecholamines because Klatt et al. (26) have presented
evidence that NO can cause an oxidative inactivation
and degradation of Iso in adipocyte cultures (26). However, there is no conclusive evidence that this process
plays any role in vivo (26).
In pioneering studies that used rats, Se deficiency
was associated with abnormal electrocardiograms (15)
and decreased basal myocardial contractility (18).
These data were questioned later because similar results could not be obtained unless a combined deficiency of Se and vitamin E were employed (36). It is of
interest that some of these authors referred to a reduced tolerance for oxygen radicals as well as some
ultrastructural alterations in isolated Langendorffperfused hearts from Se deficient but vitamin E adequate rats (47). In this study, no changes in basal atria
contractility were found due to Se deficiency. With the
use of electrically stimulated papillary muscle isolated
from rats fed a Se- and vitamin E-deficient diet, Turan
et al. (45) demonstrated that Iso-induced facilitation of
muscle contraction was smaller than in control animals although no differences were detected in the absence of Iso (i.e., basal contractions). With the use of
the same model, some underlying mechanisms were
clarified recently when a reduced adenylate cyclase
activity as well as a decreased ␤-adrenoceptor density
(⬃30%) were observed, suggesting an interference with
the ␤-adrenoceptor-adenylate cyclase coupling (39). Although Sayal et al. (39) used an experimental model
different from ours, it could nonetheless be speculated
that some of the mechanisms invoked by them might
Fig. 5. Western blot exhibits a band of ⬃130 kDa (arrow) consistent
with iNOS in heart tissue from Se⫺ mice (B). No such band is seen in
extracts of heart tissue from Se⫹ mice (A).
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In the present study, we have shown that atria taken
from mice fed a Se⫺ diet for 4 wk have a diminished
␤-adrenoceptor inotropic cardiac response (dF/dt) to
agonist stimulation (Iso or NE) when compared with
atria from mice fed the same diet supplemented with
Se (Se⫹). These data strongly relate to the diet consumed because feeding the Se⫹ diet to the Se⫺ mice for
only 1 wk before the experiment resulted in dF/dt
values similar to those observed in mice fed the Se⫹
diet throughout the entire 4-wk feeding period. The
diminished dF/dt observed in this study can be partially explained by the enhanced NOS activity detected
in atria of Se⫺ mice, together with increased serum
nitrite/nitrate levels. Furthermore, the dF/dt curves of
atria from Se⫺ mice resembled those obtained with
atria from Se⫹ mice when the experiment was conducted in the presence of L-NMMA, a nonselective NOS
blocker (27). Finally, it appears that the source of NO is
a cardiac iNOS, as shown by its selective expression
and also by the fact that the dF/dt response of Se⫺ atria
was similar to that of Se⫹ atria when the specific iNOS
inhibitor AP (13) was used.
The diminished ␤-adrenoceptor inotropic cardiac response to agonist stimulation (dF/dt) observed in this
study can be explained by the enhanced NOS activity
detected in the Se⫺ hearts and the known effects of NO
on myocardial function (28). NO stimulates myocardial
soluble guanylate cyclase to produce cGMP, which affects two major target proteins. A small increase in
cGMP levels predominantly inhibits phosphodiesterase III, whereas high cGMP levels activate cGMPdependent protein kinase. Accordingly, submicromolar
NO concentrations improve myocardial contraction,
whereas submillimolar NO concentrations decrease
contractility. However, the inotropic effects of submicromolar NO are small and probably of minor importance for myocardial contractility. In contrast, submillimolar NO concentrations produce direct inhibitory
effects of NO on ATP synthesis and voltage-gated calcium channels. Cardiodepressive actions of endogenous submillimolar NO concentrations may play a role
in certain forms of heart failure because NO reduces
the calcium affinity of the contractile apparatus, contributing to a negative inotropic effect, an abbreviation
of contraction, and an enhancement of diastolic relaxation (20). Another possibility to explain the inhibitory
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volved in increasing the expression of several cytokines, chemokines and iNOS (2, 3). The recent findings
of Prabhu et al. (34) assume importance in this connection because these workers were able to demonstrate a
NF-␬B mediated upregulation of iNOS expression in
the RAW 264.7 macrophage cell line during Se deficiency. In support of those results, macrophages from
glutathione peroxidase (GPX) knockout mice produced
more NO than those from wild-type mice (14). In addition, it has been shown that GPX can prevent apoptosis
and that NO can inhibit GPX, thereby altering the
balance between oxidants and antioxidants affecting
cellular homeostasis (1). Considering all these data
together, it is possible that the NO concentration may
be elevated in a variety of tissues and may account for
the necrotic degeneration, involving primarily the
heart muscle, but also the liver, peripheral muscle,
kidneys, pancreas, and testes observed in murine selenium and vitamin E deficiency (12).
How can the results presented here relate to human
cardiac pathophysiology? The only well-established
link between Se deficiency and human heart disease is
Keshan disease which usually presents in the form of a
DCM. It has been postulated that viral infection could
be a significant cofactor in the etiology of Keshan
disease on the basis of three lines of evidence (30).
First, coxsackie and other picornaviruses were found in
the heart tissue of affected patients. Second, Se⫺ mice
suffered a more severe myocarditis than Se⫹/⫺ mice
when inoculated with such viruses. Finally, an amyocarditic strain of coxsackie virus became myocarditic
after replication in Se⫺ mice. An abnormal immune
response after a viral-induced myocarditis has long
been postulated as a major pathogenic mechanism in
sporadic DCM.
The data presented here suggest an independent
mechanism that could also be involved because elevated NO production mediated by iNOS per se may
contribute to the myocardial impairment and elevated
apoptosis (25) associated with conditions such as myocarditis and DCM, among others. An increased expression and colocalization of tumor necrosis factor and
iNOS was observed in the myocardium of individuals
with DCM (19). It is of interest because tumor necrosis
factor production by cardiac myocytes is known to
contribute to contractile dysfunction by several mechanisms, including NO-induced myofilament desensitization (32). A major question that needs to be clarified
is the extent of Se deficiency that is required before an
increased iNOS expression is observed in the heart.
That is, is there a minimum threshold or degree of
deficiency that is needed before any increase in iNOS
expression is seen or is the elevation in iNOS expression a continuous function of Se deficiency? Whether or
not such a triggering threshold exists could have a
great impact on the role of selenium in human cardiac
health.
We thank Elvita Vannucchi and Catherine Guidry for outstanding
technical assistance.
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also be operative in our study, in addition to the involvement of NO.
The observation of iNOS expression in mouse hearts,
including those of control Se⫹ mice, is in agreement
with other studies. It has been proposed that the three
known NOS isoforms are present in the cardiomyocytes, but have distinct heterogeneous basal expression gradients, subcellular localization patterns, and
different functions (7). Low basal expression of the
iNOS has been detected in ventricular myocytes of the
ferret heart at the cytoplasmic level, a localization
conserved even after its upregulation (7). With regard
to cNOS, neuronal NOS has been recently identified in
isolated cardiac mitochondria with a proposed modulatory role for NO in oxidative phosphorylation (23), as
well as in the sarcoplasmic reticulum, where it has
been associated with Ca2⫹ release and enhanced contractility (4). eNOS localizes to caveolae (22), where
compartmentalization with ␤-adrenergic receptors and
L-type Ca2⫹ channels allows NO to inhibit ␤-adrenergic-induced inotropy (21). In addition, eNOS apparently colocalizes with the extracellular membrane
bound form of superoxide dismutase, thereby implying
a functional relationship in the modulation of the contractile apparatus (7). Considering that NO and/or
iNOS inducers have been postulated to modulate intracellular NO by a direct effect on cNOS activity (10),
it will be of future interest to investigate how such
changes may affect cardiac physiology, particularly the
role of nutritional Se status and its relationship with
NO at a molecular level, in maintaining heart health.
It should be noted that others have found that Se
deficiency may decrease serum nitrite/nitrate levels.
For example, serum nitrite/nitrate content was found
lower in male weanling C3H/HeJ mice fed a Se⫺ diet
for 5 wk than in Se⫹ controls (A. D. Smith, personal
communication). Similar results were found in Wistar
rats (35). Clearly, more studies are needed to clarify
this point.
The mechanism by which Se deficiency influences
iNOS cardiac expression is unknown. Of interest in
this context is the report that treatment of nuclear
extracts of lipopolysaccharide-activated human T cells
with relatively high concentrations of selenite inhibited nuclear factor-␬B (NF-␬B) binding and decreased
NO production (24). However, as pointed out by the
authors, this study should be interpreted with caution
because equivalent mechanisms may not be operating
in Se deficiency and in Se excess. Moreover, because of
its high chemical reactivity, the metabolism of selenite
added in vitro may not be the same as that consumed
in the diet. One possible molecular mechanism
whereby dietary Se deficiency might increase cardiac
NO output is via an activation of NF-␬B. The low level
of antioxidant selenoproteins in the Se⫺ mice would
result in an elevated oxidative stress and an increased
generation of reactive oxygen species, which in turn
would lead to an activation of NF-␬B because this
transcription factor is reactive oxygen species sensitive. The activation of NF-␬B would then cause the
upregulation of multiple genes, including those in-
SELENIUM, NITRIC OXIDE, AND HEART CONTRACTILITY
This study was supported by grants from the Universidad de
Buenos Aires, Secretaria de Ciencia y Técnica and by Proyecto de
Investgación Plurianual from the Consejo Nacional de Investigaciones Cientı́ficas y Técnicas.
22.
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