© 1999 American Heart Association, Inc.
Molecular Medicine |
Heterogeneous Basal Expression of Nitric Oxide Synthase and Superoxide Dismutase Isoforms in Mammalian Heart
Implications for Mechanisms Governing Indirect and Direct Nitric Oxide–Related Effects
From the Departments of Medicine and Pharmacology (M.V.B.), Duke University Medical Center, Durham, NC, and Department of Physiology and Biophysics (D.L.C.), State University of New York at Buffalo, NY.
Correspondence to Donald L. Campbell, Department of Physiology and Biophysics, State University of New York at Buffalo, 124 Sherman Hall, Buffalo, NY 14214. E-mail dc25{at}acsu.buffalo.edu
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Abstract—The basal expression patterns of NO synthase (NOS; endothelial [eNOS], neuronal [nNOS], and cytokine-inducible [iNOS]) and superoxide dismutase (SOD; extracellular membrane bound [ECSOD], MnSOD, and CuZnSOD) isoforms in ferret heart (tissue sections and isolated myocytes) were determined by immunofluorescent localization. We demonstrate the following for the first time in the mammalian heart: (1) heterogeneous expression patterns of the 3 NOS and 3 SOD isoforms among different tissue and myocyte types; (2) colocalization of eNOS and ECSOD at both the tissue and myocyte levels; (3) a significant gradient of eNOS and ECSOD expression across the left ventricular (LV) wall, with both enzymes being highly expressed and colocalized in LV epicardial myocytes but markedly reduced in LV endocardial myocytes; and (4) specific subcellular localization patterns of eNOS and the 3 SOD isoforms. In particular, eNOS and ECSOD are demonstrated (electron and confocal microscopy) to be specifically localized to the sarcolemma of ventricular myocytes. Similar heterogeneous eNOS and ECSOD expression patterns were also obtained in human LV tissue sections, underscoring the general importance of these novel findings. Our data suggest a strong functional correlation between the activities of sarcolemmally localized myocyte eNOS and ECSOD in governing NO·/O2- interactions and suggest that NO-related modulatory effects on cardiac myocyte protein and/or ion channel function may be significantly more complex than is presently believed.
Key Words: nitric oxide • nitric oxide synthase • superoxide dismutase • immunofluorescence • protein and ion channel modulation
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Introduction |
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The modulatory effects of nitrogen oxides on cardiac function have recently received extensive experimental attention. However, although there is general consensus that NO-related activity can exert significant chronotropic and inotropic effects on the heart,1 2 both positive and negative effects have been reported.3 4 5 6 7 8 9 10 11 12 13 Controversy also exists on the obligatory versus secondary involvement of NO-related activity on cardiac myocyte ion channel function.8 9 10 11 12 13 14 15 16 The following 2 interrelated issues may underlie the apparently conflicting results that have been reported: (1) NO synthase (NOS) protein expression patterns among different cardiac tissues and myocyte types have not been adequately analyzed, and (2) nearly all studies have equated "NO" to free radical nitric oxide, NO·, and have attributed all NO-related effects to the indirect NO·
soluble guanylyl
cyclasecyclic GMP cascade (reviewed in Reference 1717 ). In particular,
although it has been established that the indirect NO·-cGMP pathway
exists in many cardiac myocyte types,1 2 7 8 9 10 11 17 N-oxides
can also exist in other physiologically
relevant redox-related forms, including S-nitrosothiols
(RSNOs) and peroxynitrite (OONO-, a potent
oxidant). These 2 NO-related species display unique reactivity profiles
with different target molecules18 19 20 21 22 and can
potentially exert direct cGMP-independent effects through reactions
involving (1) S-nitrosylation of single regulatory thiols
(Equation 1) and/or (2) oxidation of vicinal thiols (Equation 2), as
follows: (1) RSNO+R'SHR'SNO (S-nitrosylation, single regulatory thiols) (2) RSNO/OONO-+SH+S-S-S (oxidation, regulatory disulfide formation)
Evidence for direct NO-related modulation of various ion
channel proteins has recently been reported, including aortic
calcium-activated potassium channels,23 neuronal
NMDA receptors,24 cardiac sarcoplasmic reticulum calcium
release channels,25 rabbit cloned cardiac L-type calcium
channel 
1C subunits,26 and native
L-type calcium channel complexes in ferret right
ventricular myocytes.7
A scenario therefore emerges wherein NO-related activity could modulate cardiac function by both indirect and direct mechanisms.7 18 19 20 21 26 However, the molecular mechanisms determining the predominance of these 2 pathways in cardiac myocytes are essentially unknown. Two mechanisms could be as follows:
(1) If NO/RSNOs are mainly produced by endogenous intracellular NOS activity in myocytes, the indirect effects of cGMP may dominate (eg, References 77 –11); alternatively, if NO·/O2- or RSNOs are generated either at the sarcolemmal surface or from other extracellular (ie, nonmyocyte) sources, direct NO+ transfer (Equation 1) and/or oxidation (Equation 2) reactions may dominate (eg, References 7, 187 18 –21, 23, 24, 26).
(2) OONO- forms from the rapid reaction (4 to 7x109/M-sec) of NO· with superoxide free radical, O2-.18 22 27 O2- is generated under normal physiological conditions by numerous metabolic processes, including the activity of NOS isoforms and membrane-bound NADPH oxidases.28 29 O2- could serve as a primary "scavenger" of NO· in extracellular and sarcolemmal domains, and as a result OONO- production could predominate when NO- and O2-related species are formed simultaneously in closely adjacent cellular domains.19 22 27 Hence, the expression levels and specific localization patterns of superoxide dismutase (SOD) isoforms30 31 (see Materials and Methods) could be important factors in governing NO-related effects. For example, regulation of extracellular O2- levels by the extracellular/membrane-bound isoform7 27 30 31 (ECSOD) could facilitate indirect effects; in contrast, in tissue regions where ECSOD is reduced or absent, direct NO-related effects could potentially dominate.
Although endothelial NOS (eNOS),32 33 34 35 36 cytokine-inducible NOS (iNOS),33 37 38 and neuronal NOS (nNOS)39 expression in cardiac muscle have all been reported, it has not been determined whether NOS isoform expression levels are uniform or heterogeneous among different cardiac tissue and myocyte types. Furthermore, essentially no data exist on expression patterns of SOD isoforms27 30 31 in cardiac tissue. To begin to address these important issues, we have conducted immunolocalization studies on the basal expression levels of NOS and SOD isoforms in ferret and human heart tissue sections and ferret enzymatically isolated cardiac myocytes. We concentrated on the following 2 basic questions: (1) Are basal NOS and SOD isoform expression patterns uniform or heterogeneous in the heart? (2) Are the expression patterns of NOS and SOD isoforms, and in particular eNOS and ECSOD, appropriate to suggest a functional correlation between the activities of these 2 enzyme systems7 27 in NO-related modulation of cardiac myocyte protein and ion channel function7 19 23 24 25 26 27 ?
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Materials and Methods |
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Terminology and Abbreviations
Three isoforms of NOS have been described in mammalian cells: nNOS (type I), iNOS (type II), and eNOS (type III).33 eNOS has been proposed to be associated with cell membrane caveolae,32 whereas nNOS and iNOS are assumed to be cytosolic33 39 (however, see Reference 4040 ). The following 3 isoforms of SOD have also been described in mammalian cells: MnSOD, localized to the mitochondrial matrix; CuZnSOD, diffusely spread throughout the cytoplasm; and ECSOD, distributed on cell membranes and in extracellular fluids because of the presence of a heparin binding domain.27 30 31 We have retained this terminology. For ease of presentation, the following abbreviations are used: SAN, sinoatrial node; RA, right atrium; RV, right ventricle; LV, left ventricle; and LV epi and LV endo, left ventricular epicardium and endocardium, respectively. The following terms have also been used: (1) "whole free wall" refers to a preparation obtained from the entire wall of a specific anatomic region (RA, RV, or LV), in contrast to "LV epi" and "LV endo," which refer to preparations obtained specifically from the LV epicardial and endocardial surfaces; (2) "indirect IF" refers to immunofluorescence (IF) obtained using unconjugated primary antibody and fluorochrome-conjugated secondary antibody; and (3) "direct IF" refers to results obtained by direct binding of fluorochrome-conjugated primary antibody in situ.41 42
This investigation conforms with the guidelines for the use and care of laboratory animals as outlined by the National Institutes of Health (NIH publication 8523).
Light Microscopy and Immunolocalization
Isolated Myocytes
Myocytes were enzymatically isolated from specific anatomic
regions (SAN, RA, RV [entire free wall], and approximately the middle
one third of the LV epi and LV endo surfaces) of the hearts of healthy
adult male ferrets (anesthetized via 35 mg/kg pentobarbital,
IP) using an enzyme perfusion solution (collagenase,
elastase, and protease; Langendorff apparatus) and
subsequent enzyme treatment of dissected tissue samples as previously
described.7 42 After isolation, myocytes were washed twice
in PBS (in mmol/L: NaCl 128, KCl 2,
Na2HPO4 8, and
KH2PO4 2, pH 7.2) and then
fixed as previously described.41 42 Primary antibodies and
respective dilutions used in this study were the following: (1) NOS
antibodies (anti-human mouse monoclonal antibodies [Transduction
Laboratories]), dilution 1:2500, and (2) SOD antibodies (antihuman
rabbit polyclonal antibodies [kindly provided by Drs James Crapo and
Tim Oury, Duke University]), which were ECSOD, dilution 1:2200; MnSOD,
dilution 1:2600; and CuZnSOD, dilution 1:3000. Anti-mouse IgG
conjugated with TRITC and anti-rabbit IgG conjugated with FITC (The
Jackson Laboratories) were used as secondary antibodies. For direct IF,
primary antibodies were conjugated with either TRITC (for NOS) or FITC
(for SOD). For positive controls, cardiac-specific troponin antibody
(troponin C-FITC, Novacastra Laboratories, Ltd) was used. For
NOS-negative controls, myocytes were incubated with blocking buffer,
whereas for ECSOD-negative controls myocytes were incubated with 100
units of heparin (1 hour, room temperature) before fixation. Slides
were scanned using a Zeiss LSM410 confocal microscope. Digitized images
were subsequently processed as previously described.41 42
Approximate percentage expression levels of different isoforms among
myocyte samples (Figure 5
) were estimated by visual examination
and enumeration as previously described.41 42
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Tissue Sections
Ferret Heart
After Langendorff perfusion with PBS, whole hearts were fixed as
previously described.41 42 Tissue section localizations
were performed on hearts from 4 to 7 ferrets for each isoform
analyzed. For each individual heart, a total of 40 to 50
cryosections were made of a specific anatomic region (RA, entire
ventricle), and every ninth section was sampled and analyzed.
For the ventricular sections (7 µm thickness; cut
orientation as indicated in Figure 2A), the following numbers of
measurements were performed for each isoform: eNOS and ECSOD, 7 hearts,
5 sections per heart; nNOS, 5 hearts, 4 sections per heart; iNOS, 4
hearts, 4 sections per heart; and MnSOD and CuZnSOD, 6 hearts, 4
sections per heart. eNOS and ECSOD localization in transverse
cryosections of RA (6 µm thickness; cut orientation through the
superior vena caval and sinoatrial nodal regions as shown in Figure 3A2) were conducted on 4 hearts, with 4 sections per heart. RA
and sagittal ventricular cryosections were
postfixed41 42 before immunofluorescent
localization was performed.
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Human LV
LV epi and LV endo tissue sections were prepared from 3 hearts
obtained after rapid autopsy (samples kindly provided by Dr J.S.
Reimer, Department of Pathology and Alzheimers Research Study
Group, Duke University). Cryosections (n=40 to 50) were prepared from
the indicated LV epi and LV endo regions shown in Figure 4A2
(white boxes). Every 7th section (a total of 5 sections per heart) was
sampled and analyzed for eNOS and ECSOD expression. Tissue
sections were obtained from the hearts of white males 32, 48, and 67
years of age, none of whom had any reported clinical history of heart
disease. Cryosections were rapidly fixed (3%
paraformaldehyde/1% glutaraldehyde)
and subsequently prepared and analyzed using eNOS and ECSOD
antibodies as described above for ferret heart.
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Electron Microscopy (EM)
For EM analysis of SOD isoforms in ferret LV epi and LV
endo tissue sections (Figure 6), standard double immunolabeling
techniques were applied.43 44 45 Ultrathin (60 nm)
cryosections were blocked with preimmune serum followed by primary
antibody incubation. Binding patterns were determined by application of
protein A gold (PAG; CuZnSOD, 5-nm particles; MnSOD, 10-nm particles;
and ECSOD, 15-nm particles). Sections were then analyzed using
a Philips CM-10 electron microscope.
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Antibody Characterization
Membrane Preparations
Ferret hearts were rinsed in PBS at room temperature and then
frozen at –70°C. Both ferret heart and various standard membrane
preparations (see below) were prepared as previously
described,46 with the following modifications (all
procedures conducted at 4°C). Solutions contained the following
mixture of protease inhibitors: aprotinin 2 mg/mL;
benzamide 0.5 mmol/L; iodoacetamide 0.6 mmol/L; leupeptin
0.15 µmol/L; pefabloc 0.5 mmol/L; pepstatin 1
µmol/L; and 1,10 phenanthroline 0.5 mmol/L. Specific
tissue samples were suspended in TE buffer (10 mmol/L TRIS and
1 mmol/L EDTA), homogenized, centrifuged
(1000g, 10 minutes), and the supernatant was collected and
recentrifuged (60 000g, 30 minutes). The pellet was
then resuspended in TE buffer with 0.6 mol/L KI, incubated on ice for
15 minutes, centrifuged (60 000g, 30 minutes), and
washed twice with TE buffer to remove KI. The pellet was solubilized in
2% Triton x100–TE buffer on ice for 1 hour and then
centrifuged at 15 000g for 15 minutes. Protein
content was determined using a BCA protein assay.
Immunoblot Analysis
Membrane and tissue homogenates were resolved on
7.5% or 10% SDS-PAGE gels (Prosieve 50 solution, FMC) with
appropriate protein markers, followed by transfer to ECL Hybond
nitrocellulose membrane (Amersham). After incubation with 1% BSA in
PBS–0.05% Tween 20 (blocking buffer), the blots were incubated in
appropriate antibody diluted in blocking buffer (dilutions were the
following: eNOS, nNOS, and iNOS, 1:2500; ECSOD, 1:2500; MnSOD, 1:2600;
and CuZnSOD, 1:3000) for 1 hour, washed 3 times with PBS–Tween 20, and
subsequently incubated in horseradish peroxidase–conjugated secondary
antibody. The blots were washed 5 times and visualized by enhanced
chemiluminescence (Amersham). Relative band intensities were compared
by use of NIH Image, IP Laboratory Gel, and Image Quant software. For
each antibody, a specific "positive control" was used, for which
that antibody reacted at a maximal level, and the band intensities
obtained from these positive controls were defined as 100%. The
density of each preparation was then percentage-normalized relative to
the appropriate 100% positive control. Positive controls were as
follows: eNOS, human endothelial cells (En); nNOS, rat
pituitary cells (Pi); iNOS, mouse macrophages (Ma); ECSOD and
MnSOD, human lung cells (HL); and CuZnSOD, human red blood cells. We
wish to emphasize that the relative percentage band intensities
reported (Figure 1) are given only for comparative purposes for
each individual antibody and are not for comparative purposes between
different antibodies.
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Interpretive Limitations
In interpreting our IF results, we wish to emphasize the
following 2 points. (1) For numerous reasons (eg, monoclonal versus
polyclonal antibodies, possible differences in number, accessibility,
and/or affinity of epitopes, etc), the fluorescent intensities
obtained for any one specific NOS or SOD isoform cannot be
quantitatively compared with those obtained from another isoform; ie,
only relative and/or qualitative comparisons among different isoforms
are valid. Therefore, for comparative purposes, the relative intensity
profiles42 of different isoforms shown (Figures 2
and 3) were each separately normalized to the maximum
fluorescence obtained for each isoform (ie, maximum relative
intensity, 100%). (2) IF results obtained on isolated myocyte samples
(Figures 5 and 6) specifically measure NOS and SOD
isoform protein expression within myocytes. However, in intact tissue
sections (Figures 2, 3, and 4), NOS and/or SOD
isoforms may not be expressed only in myocytes but also within other
nonmyocyte cell types, eg, endothelial cells,
smooth muscle cells, and neurons. Therefore, both the overall IF
patterns and relative intensity profiles obtained from the
ventricular and RA tissue sections cannot at present be
attributed exclusively to NOS and SOD isoform expression within
myocytes.
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Results |
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Antibody Specificity: Immunoblot Analysis
Representative immunoblot results on the specificity of the antibodies used are shown in Figure 1
. Results were obtained from
protein homogenates prepared from specific regions of
ferret heart (RV and LV whole free walls, LV epi, and LV endo) and
brain (Br) and various positive control tissue preparations (eNOS,
human En; nNOS, rat Pi; iNOS, mouse Ma; ECSOD and MnSOD, HL; and
CuZnSOD, human red blood cells [RBC]; see Materials and Methods:
Immunoblot Analysis). Band intensities were expressed as percentage
intensity relative to indicated specific positive controls. All
3 NOS antibodies displayed prominent single binding bands in both the
various positive controls and ferret heart preparations. Among the
heart preparations, eNOS (
140 kDa) was prominent in RV (relative
band intensity, 82% of En) and LV (whole free wall, 81% of En);
however, when LV epi and LV endo preparations were tested separately,
eNOS in LV endo was markedly reduced (17% of En) compared with that in
LV epi (69% of En). Both nNOS (150 to 160 kDa) and iNOS (130
kDa) could also be detected at low basal levels in both RV (nNOS, 21%
of Pi; iNOS, 9% of Ma) and LV (nNOS, 16% of Pi; iNOS, 7% of Ma).
Among the SOD antibodies, in general multiple binding patterns were
observed in all preparations analyzed (MnSOD prominent at 22
kDa, CuZnSOD at 25 and 29 kDa, ECSOD prominent at 33 kDa and
more weakly at 29 kDa). These SOD antibody binding results are
consistent with the known (but presently incompletely
characterized) multimeric structure of SOD isoforms in other
noncardiac cell types.27 30 31 The SOD band patterns were
also very similar among the various positive control and ferret cardiac
preparations. However, when LV epi and LV endo preparations were tested
separately, the prominent band for ECSOD binding (33 kDa) was
markedly reduced in LV endo (relative intensity of 23% of HL) compared
with LV epi (63% of HL). Similar immunoblot results were
consistently obtained from a total of 4 whole LV preparations
and 6 LV epi and 6 LV endo preparations (each preparation made from a
different heart).
Ferret Heart: NOS and SOD Isoform Protein Expression Patterns in
Tissue Sections
Our immunoblot results (Figure 1) suggested
that there may be differences in expression levels of NOS and SOD
isoforms in different regions of the heart, particularly across the LV
wall. Therefore, we next determined overall expression patterns of NOS
and SOD isoforms in ferret heart tissue sections. We particularly
concentrated on eNOS and ECSOD because of the roles that these 2
enzymes could play in indirect versus direct extracellular/sarcolemmal
NO-related effects.7 19 27
eNOS and ECSOD: Ventricular and Right Atrial
Tissue Sections
Figure 2 shows IF results obtained
on eNOS and ECSOD expression patterns in adjacent sagittal sections
(7 µm thick) of the ferret ventricle (see Figure 2A and 2B for section orientation). To verify secondary antibody specificity,
a series of positive and negative control measurements were initially
conducted. Binding of cardiac-specific troponin antibody (positive
control) gave a high fluorescence signal that was uniform
across all ventricular regions (Figure 2B). In
contrast, in the absence of primary eNOS antibody (Figure 2C)
and heparin-exposed sections treated with primary ECSOD antibody
(Figure 2D), application of secondary antibodies (negative
controls) failed to produce any significant fluorescence.
In marked contrast to troponin, binding of both eNOS (Figure 2E, main panel, red fluorescence) and ECSOD (Figure 2F, main
panel, green) antibodies gave rise to distinct and
heterogeneous basal expression patterns among different
regions of the ventricle. Both eNOS and ECSOD expression levels were
relatively high and uniform across the entire free wall of the RV, high
in the apical and midventricular regions of the LV epi, and
markedly reduced or absent in the LV endo and the LV side of the
septum. This heterogeneous expression pattern is further
emphasized in Figure 2E1 through 2E4 and 2F1 through 2F4, which
show x60 enlargements of the tissue regions indicated by the white
boxes.
Comparison of the adjacent sections shown in Figure 2E and 2F
suggested that there may also be a marked colocalization of eNOS and
ECSOD expression in intact ventricular tissue. To verify
colocalization, direct IF measurements (see Materials and Methods) were
conducted on single sagittal sections treated with both eNOS and ECSOD
antibodies. As shown in Figure 2G, both eNOS and ECSOD
expression were found to be highly colocalized (ie, yellow-orange). To
further demonstrate heterogeneous expression and
colocalization, the relative fluorescence intensity
profiles42 of eNOS and ECSOD were measured transversely
at the indicated apical, midventricular, and basal regions
(cyan lines 5, 6, and 7 in Figure 2E and 2F). Although at
present we do not wish to impart any further quantitative
interpretation of these data (see Materials and Methods: Interpretive
Limitations), it is nonetheless clear that there was a striking
similarity between the relative intensity profiles of eNOS (Figure 2E5 through 2E7) and ECSOD (Figure 2F5 through 2F7) among
corresponding transverse regions of the 2 adjacent sections. This is
further emphasized in Figure 2G5 through 2G7, which overlay the
relative eNOS and ECSOD intensity profiles for comparative purposes. In
summary, heterogeneous eNOS and ECSOD expression and
colocalization patterns similar to those shown in Figure 2 were
consistently observed in ventricular sagittal
sections prepared from a total of 7 ferret hearts.
Representative eNOS and ECSOD expression patterns in
ferret right atrial tissue sections (6 µm thick) are shown in
Figure 3A through 3D (see figure legend
for details). In contrast to the ventricle, both eNOS and ECSOD were
uniformly expressed (Figure 3B and 3C; indirect IF) and
colocalized (Figure 3D; direct IF; colocalization is indicated
by bright yellow) among all of the RA pectinate myocyte types examined
from the indicated RA regions. Similar results were obtained from RA
sections prepared from a total of 4 hearts.
Additional IF measurements (data not shown) indicated that eNOS and ECSOD expression was high and uniform in ferret SAN sections. These results indicate that at the tissue level, eNOS and ECSOD expression patterns vary not only among the major anatomic regions of the ferret heart (SAN/atrium versus ventricle) but also within specific anatomic regions of the ventricle (RV/LV epi versus LV endo/septum).
nNOS, iNOS, MnSOD, and CuZnSOD: Ventricular Tissue
Sections
Having demonstrated heterogeneous expression and
colocalization of eNOS and ECSOD in the ventricle, we next sought to
determine whether there was basal expression of nNOS, iNOS, MnSOD, and
CuZnSOD isoforms in the ventricle and whether the basal expression
patterns of these other isoforms were uniform or
heterogeneous. Representative results on
basal expression patterns of these specific isoforms in ferret
ventricular sagittal tissue sections are shown in Figure 3E through 3J (for comparative purposes, the relative intensity
profiles [see Materials and Methods] of each isoform across the same
transverse midventricular region are also shown in Figure 3E1 through 3J1). With regard to NOS isoforms, 2 interesting
findings were as follows: (1) consistent with the
immunoblot results (Figure 1), low basal expression
patterns of both nNOS39 and iNOS47 could be
detected at the intact ventricular tissue level (Figure 3F and 3G), and (2) the overall expression patterns for both
nNOS and (basal) iNOS were markedly different from that of eNOS
(compare Figure 3E with 3F and 3G). In particular, nNOS
expression was an approximate inverse of eNOS expression, being
relatively high in LV endo and septum and low to absent in LV epi and
RV. With regard to SOD isoforms, both MnSOD and CuZnSOD could
also be detected (Figure 3I and 3J). However, in contrast to
ECSOD, the expression of these 2 isoforms was relatively more uniform
across all ventricular regions (compare Figure 3H
with 3I and 3J; see also Discussion). In summary, similar expression
patterns for nNOS, iNOS, MnSOD, and CuZnSOD were observed in
ventricular sagittal sections prepared from totals of 5
hearts (nNOS), 4 hearts (iNOS), and 6 hearts (MnSOD and CuZnSOD).
Human Heart: eNOS and ECSOD Expression Patterns in LV Epi and LV
Endo Tissue Sections
To test for the possibility that heterogeneous
expression of eNOS and ECSOD may be a unique characteristic of ferret
heart, we also conducted a limited IF analysis of eNOS and
ECSOD expression in LV epi and LV endo tissue samples obtained from
human hearts (see Materials and Methods). Figure 4A through 4E shows
representative IF results obtained from cross-sectional
tissue samples of the human LV (see Figure 4A1 and 4A2 for
orientation and figure legend for details). For comparative purposes,
Figure 4F through 4J shows results on eNOS and ECSOD expression
obtained from a similar cross-sectional sampling of the ferret
ventricle. Comparison of the IF results from the 2 preparations
indicated a very similar overall tissue expression pattern, ie, in
human LV tissue there was also a marked heterogeneous
expression of eNOS and ECSOD, with expression of both being high in LV
epi (Figure 4B and 4C) and reduced in LV endo (Figure 4D and 4E). Similar results from paired LV epi and LV endo tissue sections
were obtained from 3 human hearts.
Ferret Heart: Isolated Myocyte Analysis
Having determined overall NOS and SOD isoform expression levels in
ferret cardiac tissues, we next conducted indirect IF measurements on
samples of myocytes41 42 enzymatically isolated from
ferret SAN, RA (entire free wall), RV (entire free wall), and LV epi
and LV endo regions. Selected positive IF results for NOS and SOD
isoform expression in the 4 working myocyte types are shown in Figure 5A through 5D, whereas summarized
mean results on approximate percentage myocyte expression of NOS and
SOD isoforms in all 5 myocyte types are given in Figure 5E and 5F.
With regard to NOS isoforms (Figure 5E), eNOS was the
predominantly expressed isoform and was observed in the majority of SAN
and RA myocytes; however, among ventricular myocytes, eNOS
expression was highest in LV epi myocytes (89.1±0.1%), intermediate
in RV myocytes (53.3±0.5%), and markedly reduced in LV endo myocytes
(29.9±0.5%). A detectable nNOS39 and basal iNOS
expression47 was also observed in a small subpopulation of
each myocyte type, with expression of both being highest in LV endo
myocytes (20%) and lower to essentially absent in the other myocyte
types. In addition, exposure of all 5 isolated myocyte types to
selected cytokines (tumor necrosis factor- and
interferon-
; 1- to 4-hour incubations in short-term culture, 37°C)
led to a significant increase in iNOS expression, further corroborating
both specific iNOS expression and antibody specificity (data not
shown).
With regard to SOD isoforms (Figure 5F), ECSOD was expressed in
the majority of SAN and RA myocytes, but displayed a marked
heterogeneous expression pattern among
ventricular myocytes, being highest in LV epi myocytes
(85.7±0.9%), intermediate in RV myocytes (57.0±0.8%), and lowest in
LV endo myocytes (45.1±1.9%). In contrast, MnSOD was nearly uniformly
expressed in the majority of all 5 myocyte types. CuZnSOD was also
nearly uniformly expressed in all myocyte types except for RV myocytes,
in which it was reduced.
In summary, at the level of isolated LV epi and LV endo myocytes, eNOS and ECSOD expression paralleled each other. These results indicate that heterogeneous eNOS and ECSOD expression patterns in LV epi versus LV endo myocytes are contributing to the net expression gradients observed in the intact LV wall. These same general conclusions also apply to our results on nNOS and basal iNOS expression.
Ferret Heart: Subcellular Localization of eNOS and SOD
Isoforms
To determine the subcellular localization patterns of SOD isoforms
within ventricular myocytes, we used EM. Figure 6A1 through 6A4 shows EM results on the
localization patterns of SOD isoforms in 60 nm thick sections obtained
from ferret LV epi (Figure 6A1 through 6A3) and LV endo (Figure 6A4) tissue. In LV epi myocytes, MnSOD specifically localized to
the mitochondria (Figure 6A1), CuZnSOD was uniformly distributed
throughout the myoplasm but was excluded from the mitochondria and
sarcolemma (Figure 6A2), and ECSOD was highly expressed and
specifically localized to the sarcolemma (Figure 6A3). In LV
endo myocytes (Figure 6A4), a similar SOD isoform expression
pattern was also observed; however, although ECSOD was again
specifically localized to the sarcolemma, its density was markedly
reduced compared with that of LV epi myocytes (compare Figure 6A3 and 6A4). Similar SOD isoform expression patterns were
consistently observed in LV epi and LV endo sections prepared
from 3 ferret hearts.
EM localization of NOS isoforms was hampered because the NOS antibodies
used failed to significantly bind protein A gold. However, because
ECSOD is specifically localized to the sarcolemma, ECSOD
fluorescence patterns can serve as a specific sarcolemmal
marker. Therefore, to determine localization of eNOS, we conducted
confocal microscopic analysis42 on ferret isolated
myocytes that had been double labeled (direct IF) for both eNOS and
ECSOD. Representative results obtained from RV, LV epi,
and LV endo myocytes are shown in Figure 6B through 6D. When a
series of optical Z sections (0.75 µm thickness) were made
progressively through the width of all myocyte types analyzed,
the fluorescence intensity patterns were localized to the outer
sarcolemmal regions. These results indicate that eNOS is specifically
localized to the sarcolemma. It should also be noted that the eNOS and
ECSOD fluorescence intensities were relatively more prominent
in the RV and LV epi myocytes (Figure 6B and 6D) compared with
the LV endo myocytes (Figure 6C).
In summary, these EM and confocal Z-section results demonstrate that eNOS and ECSOD are localized to the sarcolemma of ventricular myocytes and directly confirm the heterogeneous eNOS/ECSOD expression gradients in LV epi versus LV endo myocytes.
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Discussion |
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Novel Results: Heterogeneous NOS and SOD Isoform Expression in Heart
It is now recognized that ion channel expression in the heart can be quite heterogeneous.41 42 Hence, enzyme systems involved in modulation of cardiac myocyte ion channel function will also probably not be uniformly expressed in the heart, a concept generally ignored in considerations of cardiac function. Our results are therefore important in that they are the first demonstration of distinct heterogeneous basal expression gradients and subcellular localization patterns of NOS and SOD isoforms in the heart. These novel results may provide new insights into mechanisms governing NO-related modulation of cardiac myocyte protein function and may have direct relevance to present controversies regarding NO-related effects on cardiac myocyte ion channels.
eNOS and ECSOD: General Implications
Possibly our most significant finding is the expression gradient
and colocalization of eNOS and ECSOD isoforms across the LV wall of
both the ferret and human heart (Figure 4). The fact that
heterogeneous eNOS/ECSOD expression exists in both ferret
and human heart underscores the general significance of this finding.
Our results on ferret isolated myocytes further demonstrate that eNOS
and ECSOD expression is markedly heterogeneous among LV epi
versus LV endo myocytes and is specifically localized to the sarcolemma
of ventricular myocytes (although our present data do
not allow us to conclude that eNOS and ECSOD are tightly colocalized to
the same specific sarcolemmal microdomains, eg,
caveolae).32 These findings strongly imply a functional
correlation between the activities of endogenous myocyte
eNOS and ECSOD in the regulation of extracellular and/or sarcolemmal
NO·/O2-
interactions.7 19 27 As a result, there may be
significant differences in indirect versus direct NO-related protein
and/or ion channel responses between different cardiac myocyte types.
For example, in LV epi myocytes, regulation of extracellular
O2- levels by
ECSOD27 could facilitate indirect (cGMP-dependent)
eNOS-mediated effects. In contrast, because of the markedly reduced
levels of both eNOS and ECSOD, direct NO-related effects (extracellular
OONO-, circulating RSNOs, and/or
endogenous oxidants18 19 20 21 48 49 ; Equations 1
and 2) could potentially dominate in LV endo myocytes.
eNOS and ECSOD: Implications for Myocyte Ion Channels
Our results may have implications for 2 voltage-gated ion channel
types that have recently received extensive experimental attention, the
basal L-type Ca2+ current, ICa,
L, and the rapidly inactivating and transient outward
K+ currents, IKr and
Ito, respectively.
ICa, L
The basal L-type Ca2+ current,
ICa, L, in ferret right
ventricular (RV) myocytes can be selectively modulated by
NO· (indirect cGMP-dependent inhibition) versus
RSNOs/OONO- (direct cGMP-independent
stimulation).7 Our present results therefore
provide a molecular basis for the dual-mechanism NO-related regulatory
model proposed in this previous patch-clamp study7 and the
underlying assumptions made therein regarding mechanisms governing
sarcolemmal NO·/O2-
interactions (see Figure 10 in Reference 77 ; see also Reference 2727 ).
Extrapolating from these previous results,7 our
present IF results could imply the following: (1) that LV epi
myocytes may be more influenced by endogenous myocyte eNOS
and ECSOD activity, whereas LV endo myocytes may be more influenced by
NO-related species generated by remote (nonmyocyte) cell
sources (eg, References 48 and 4948 49 ) and (2) that indirect
(inhibitory) effects on ICa, L
may be more dominant in LV epi myocytes, whereas direct (stimulatory)
effects on ICa, L may be more prevalent
in LV endo myocytes.7
In contrast to LV, the intermediate expression of eNOS and ECSOD in ferret RV myocytes suggests that there may be approximately equal NO-related indirect versus direct responses on ICa, L among the RV myocyte population. It is therefore interesting to note that in our previous study,7 we observed that the compound SIN-1 (which generates both NO· and OONO-) stimulated ICa, L in approximately half of the RV myocytes studied and inhibited ICa, L in the other half. These SIN-1 results may now be viewed as being consistent with the intermediate expression pattern of ECSOD in RV myocytes and the predictions of the proposed dual-mechanism redox-regulatory model.7 Heterogeneous ECSOD expression may also provide a basis for the multiple effects that SIN-1 exerts on ICa, L in other cardiac myocyte preparations (eg, References 5 and 505 50 ).
Heterogeneous eNOS and ECSOD expression may also provide a molecular basis for some of the conflicting results reported on the involvement of myocyte NOS activity in modulation of ICa, L. Two examples would include the following: (1) apparent differences among cardiac myocyte types in mechanisms governing amplitude and frequency-dependent characteristics of basal ICa, L7 11 51 and (2) controversy over the obligatory involvement of endogenous myocyte NO production in cholinergic-mediated inhibition of ICa, L and other ion channels.8 9 10 11 12 13 14 15 16 52 53 Our results would suggest that generalities about obligatory involvement of NO production in cholinergically mediated modulation of ICa, L may not be applicable to all cardiac myocyte types.1 11 8 9 10 11 12 13 14 15 16 52 53 We would therefore suggest in future studies of cholinergic modulation (as well as other neuromodulatory compounds) that very careful attention be paid to the exact anatomic tissue region from which myocytes are isolated (particularly ventricular epicardium versus endocardium).
IKr and
Ito
Rapidly inactivating (IKr) and
transient outward (Ito)
K+ currents are important contributors to
repolarization in many cardiac myocyte types.54
Heterogeneous expression patterns of potassium channel
subunits (ERG, Kv1.4, Kv4.2, and Kv4.3) that correlate with
IKr and the 2 major
Ito phenotypes in ferret LV epi and
LV endo myocytes have recently been
demonstrated.41 42 Interestingly, both
ERG41 and Kv4.242 localization in ferret
heart closely parallel eNOS and ECSOD expression, whereas Kv1.4 is
almost exclusively expressed in ferret LV endo (ie, where eNOS and
ECSOD expression is minimal). The latter observation is particularly
intriguing, because inactivation of heterologously expressed Kv1.4 is
redox-sensitive.55 These potassium channels may therefore
provide a selective system for regional NO-related modulation of
cardiac function potentially more complex than that suggested for
ICa, L.7
iNOS and nNOS
Whereas eNOS was found to be the predominantly expressed isoform,
our isolated myocyte results clearly indicated that both nNOS and iNOS
are basally expressed in some ferret cardiac myocyte types. The
specificity of our iNOS measurements was corroborated by the
observation that iNOS expression in ferret myocytes could be
upregulated on exposure to cytokines (data not shown). Our
finding of nNOS expression is also in general agreement with the recent
finding of nNOS expression in the sarcoplasmic reticulum of rabbit and
human myocardium.39 Intriguingly, the basal
expression of nNOS and iNOS in the ferret ventricle (Figures 3 and 5) is an approximate inverse of that of eNOS. While the
functional consequences of this are presently unclear, if nNOS and
iNOS are localized intracellularly, then their relative predominance in
LV endo myocytes may provide one mechanism for partially countering the
hypothesized predominance of direct NO-related effects in this myocyte
type.
MnSOD and CuZnSOD
Consistent with its mitochondrial localization, MnSOD was
relatively uniformly expressed in both ventricular sagittal
sections and the majority of all enzymatically isolated myocyte types
analyzed. Interestingly, myoplasmic CuZnSOD was also relatively
uniformly expressed in all myocyte types except for RV myocytes, where
it was reduced (Figure 5F). There thus appears to be a slight
discrepancy between CuZnSOD expression at the whole
ventricular tissue level (Figure 3J) versus that
observed in isolated myocytes. This was the only exception to our
general finding that SOD (and NOS) isoform expression in isolated
myocytes paralleled that observed in whole tissue sections.
Although the underlying reasons for this discrepancy are presently
unclear, it may indicate additional contributions of CuZnSOD expression
in other nonmyocyte cell types in the ventricular
sections (see Materials and Methods: Interpretive Limitations).
Potential Pathological Implications
In conclusion, our results also have some potentially important
implications for pathological conditions. For example, our results
suggest that iNOS-related pathology1 2 may be initially
more prevalent in LV endo myocytes. Possibly even more intriguing is
the fact that many of the injurious effects produced after cardiac
ischemic episodes are caused by increased
O2-
production.22 56 Because of lower levels of ECSOD,
LV endo myocytes may be much more prone to the injurious effects of
excessive O2- generation. The
higher expression levels of nNOS and iNOS in LV endo myocytes may also
exacerbate excessive OONO--mediated effects. In
this regard, it may be noted that nitrotyrosine formation (an indirect
indicator of OONO- generation) occurs around
foamy macrophages in human atherosclerotic lesions and in
myocytes obtained from hearts of patients with myocarditis or
sepsis.22 57 It will therefore be interesting to determine
whether nitrotyrosine formation is most prominent in LV endo myocytes
and whether eNOS/ECSOD expression patterns are altered in LV epi versus
LV endo tissues during or after various pathological conditions.
|
Acknowledgments |
|---|
This work was supported by National Heart, Lung, and Blood Institute Grant HL5891302 to D.L.C. We thank Dr Harold C. Strauss for his generous support and encouragement during the initial stages of this study.
|
Footnotes |
|---|
This manuscript was sent to Michael R. Rosen, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received September 23, 1998; accepted July 15, 1999.
|
References |
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G. C L Bett, S. Dai, and D. L Campbell Cholinergic modulation of the basal L-type calcium current in ferret right ventricular myocytes J. Physiol., July 1, 2002; 542(1): 107 - 117. [Abstract] [Full Text] [PDF]
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X. Wang and A. A. Abdel-Rahman Estrogen modulation of eNOS activity and its association with caveolin-3 and calmodulin in rat hearts Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2309 - H2315. [Abstract] [Full Text] [PDF]
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R. M Bell, C. C.T Smith, and D. M Yellon Nitric oxide as a mediator of delayed pharmacological (A1 receptor triggered) preconditioning; is eNOS masquerading as iNOS? Cardiovasc Res, February 1, 2002; 53(2): 405 - 413. [Abstract] [Full Text] [PDF]
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 O. Gealekman, Z. Abassi, I. Rubinstein, J. Winaver, and O. Binah Role of Myocardial Inducible Nitric Oxide Synthase in Contractile Dysfunction and {beta}-Adrenergic Hyporesponsiveness in Rats With Experimental Volume-Overload Heart Failure Circulation, January 15, 2002; 105(2): 236 - 243. [Abstract] [Full Text] [PDF]
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V. Stangl, G. Baumann, K. Stangl, and S. B Felix Negative inotropic mediators released from the heart after myocardial ischaemia-reperfusion Cardiovasc Res, January 1, 2002; 53(1): 12 - 30. [Abstract] [Full Text] [PDF]
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R. M Bell and D. M Yellon The contribution of endothelial nitric oxide synthase to early ischaemic preconditioning: the lowering of the preconditioning threshold. An investigation in eNOS knockout mice Cardiovasc Res, November 1, 2001; 52(2): 274 - 280. [Abstract] [Full Text] [PDF]
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 U. Flögel, M. W. Merx, A. Gödecke, U. K. M. Decking, and J. Schrader Myoglobin: A scavenger of bioactive NO PNAS, December 28, 2000; (2000) 11460298. [Abstract] [Full Text]
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Y.-T. Xuan, X.-L. Tang, Y. Qiu, S. Banerjee, H. Takano, H. Han, and R. Bolli Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2360 - H2371. [Abstract] [Full Text] [PDF]
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A. E Belevych and R. D Harvey Muscarinic inhibitory and stimulatory regulation of the L-type Ca2+ current is not altered in cardiac ventricular myocytes from mice lacking endothelial nitric oxide synthase J. Physiol., October 15, 2000; 528(2): 279 - 289. [Abstract] [Full Text] [PDF]
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N. Paolocci, U. E. G. Ekelund, T. Isoda, M. Ozaki, K. Vandegaer, D. Georgakopoulos, R. W. Harrison, D. A. Kass, and J. M. Hare cGMP-independent inotropic effects of nitric oxide and peroxynitrite donors: potential role for nitrosylation Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1982 - H1988. [Abstract] [Full Text] [PDF]
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U. Flogel, M. W. Merx, A. Godecke, U. K. M. Decking, and J. Schrader Myoglobin: A scavenger of bioactive NO PNAS, January 16, 2001; 98(2): 735 - 740. [Abstract] [Full Text] [PDF]
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X. Wang and A. A. Abdel-Rahman Estrogen modulation of eNOS activity and its association with caveolin-3 and calmodulin in rat hearts Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2309 - H2315. [Abstract] [Full Text] [PDF]
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J. Heger, A. Godecke, U. Flogel, M. W. Merx, A. Molojavyi, W. N. Kuhn-Velten, and J. Schrader Cardiac-Specific Overexpression of Inducible Nitric Oxide Synthase Does Not Result in Severe Cardiac Dysfunction Circ. Res., January 11, 2002; 90(1): 93 - 99. [Abstract] [Full Text] [PDF]
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