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

Mulugu V. Brahmajothi, Donald L. Campbell
https://doi.org/10.1161/01.RES.85.7.575
Circulation Research. 1999;85:575-587
Originally published October 1, 1999

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Abstract

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.

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 cyclase→cyclic 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′SH→R′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 α1C 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 7×109/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 ?

Materials and Methods

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

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 3A⇓2) were conducted on 4 hearts, with 4 sections per heart. RA and sagittal ventricular cryosections were postfixed41 42 before immunofluorescent localization was performed.

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 4A⇓2 (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.

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.

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 ×100–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.

Figure 1.
Figure 1.

Antibody specificity: immunoblot analysis. Shown are protein homogenates (20 μg protein/lane) prepared from ferret RV (entire free wall), LV (entire free wall), epicardial and endocardial surfaces of LV (LV ep, LV en), and brain (Br). HM and LM indicate high and low molecular mass markers. Positive controls as described in Materials and Methods. eNOS: human En (positive control) and ferret RV, LV, and Br homogenates give eNOS antibody binding at ≈140 kDa. Note that there is stronger binding in LV epi compared with LV endo. nNOS: rat Pi (positive control) and ferret Br show strong nNOS antibody binding (≈150 to 160 kDa), whereas ferret RV and LV bind faintly. iNOS: mouse Ma (positive control) shows strong iNOS antibody binding (≈130 kDa), whereas ferret RV, LV, and Br show weak to very faint binding. ECSOD: HL (positive control) and ferret RV, LV, and Br show 1 distinct band (≈33 kDa) and 1 weaker band (≈29 kDa). Note that there is stronger binding to LV epi than to LV endo. MnSOD: HL (positive control) and ferret RV, LV, and Br all display prominent binding to MnSOD antibody at ≈22 kDa. CuZnSOD: human red blood cells (RBC; positive control) and ferret RV, LV, and Br show binding to CuZnSOD antibody at ≈25 and ≈29 kDa.

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.

Figure 2.
Figure 2.

eNOS and ECSOD expression in ferret ventricular sagittal tissue sections. A, Section orientation: whole ferret heart sagittally bisected. Note that in this and all subsequent ventricular sections, the atria have been removed. B through G, IF results on ventricular sagittal sections (each 7 μm thick). B through D, Positive and negative IF controls. B, Section orientation and positive control: cardiac-specific troponin primary antibody gave a high and uniform fluorescence in all regions (direct IF). AO indicates aorta; CA, coronary artery; and Sep, septum. C and D, Negative controls: lack of significant fluorescence after application of appropriate secondary antibodies in the absence of primary eNOS antibody (C); section absorbed with heparin and subsequently tested with ECSOD antibody (D). E through G, IF results from adjacent 7 μm thick sections. Main panels E and F, eNOS (red; E) and ECSOD (green; F) expression, indirect IF. Main panel G, Colocalization of eNOS and ECSOD (ie, yellow-orange) in a single section, double direct IF with eNOS and ECSOD antibodies. Subpanels E1 through E4, F1 through F4, and G1 through G4, ×60 enlargements taken from the indicated (white boxes) apical and midventricular LV epi and LV endo regions in panels E, F, and G, respectively. Calibration bars: E1 and E3=10 μm. Subpanels E5 through E7, F5 through F7, and G5 through G7, Relative intensity profiles for eNOS and ECSOD measured at the indicated basal (5), midventricular (6), and apical regions (7) (cyan transverse lines) for the corresponding sagittal sections in panels E, F, and G, respectively. Relative intensities were normalized by defining 0% as minimal fluorescence measured in negative controls (eg, panel C), and 100% as maximum fluorescence measured for each specific antibody at the 3 indicated cyan lines (see Material and Methods: Interpretive Limitations).

Figure 3.
Figure 3.

A through D, eNOS and ECSOD expression in ferret right atrial (RA) tissue sections. A1 and A2, Section orientation: upper (basal) view of intact ferret heart with RA present (A1). SVC and IVC indicate superior and inferior vena cava, respectively. Dotted yellow and black line indicates the incision. A2, Cut-open RA preparation. Pink line indicates the region from which sections in panels B through D were taken; black box indicates the SA node region. CT indicates crista terminalis. Main panels B, C, and D, eNOS (red; B) and ECSOD (green; C) expression in adjacent RA sections, indirect IF; eNOS and ECSOD colocalization is shown (D; yellow-orange) in an immediately adjacent RA section (double direct IF). Subpanels B1 through B3, C1 through C3, D1 through D3, ×60 enlargements taken from indicated (white boxes) RA regions in panels B, C, and D, respectively. Calibration bars: B1, C1, and D1=15 μm. E through J, eNOS (E), nNOS (F), iNOS (G), ECSOD (H), MnSOD (I), and CuZnSOD (J) expression in ferret ventricular sagittal tissue sections (7 μm thickness, adjacent sections shown for each NOS and SOD isoform). Relative intensity profiles for each isoform at the indicated midventricular cross sections (cyan lines) are also shown in the corresponding adjacent panels, E1 through J1.

Figure 4.
Figure 4.

Comparison of eNOS and ECSOD expression in cross sections of human and ferret LV tissue. A through E, eNOS and ECSOD expression in human LV epi and LV endo tissue sections. Representative indirect IF results obtained from the heart of a 32-year-old male (see Materials and Methods: Tissue Sections). A1 and A2, Section orientation: whole heart (A1; line indicates transverse cut orientation) and whole heart transversely bisected (A2) showing specific LV epi and LV endo regions (white boxes indicate ×60 enlargements) from which sections were prepared. B through E, eNOS and ECSOD expression in LV epi and LV endo sections (7 μm thick) from areas indicated in panel A2. Calibration bar: B=10 μm. F through J, Comparative results on eNOS and ECSOD expression in a cross section of the whole ferret heart. F1, Orientation: schematic representation of ferret heart. Cut orientation is indicated by white line. F2, Positive control: whole cross section of ferret heart tested with anti-troponin (TnIc) antibody. Note the uniformity of fluorescence in all regions. White boxes (×60 enlargements) indicate the regions where the cross sections shown in panels G through J were taken. G through J, eNOS and ECSOD expression in ferret LV epi and LV endo cross-sectional tissue sections. Fluorescence patterns were obtained from indicated white boxes shown in panel F2.

Results

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 2E⇑1 through 2E4 and 2F1 through 2F4, which show ×60 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 2E⇑5 through 2E7) and ECSOD (Figure 2F⇑5 through 2F7) among corresponding transverse regions of the 2 adjacent sections. This is further emphasized in Figure 2G⇑5 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 3E⇑1 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 4A⇑1 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⇓.

Figure 5.
Figure 5.

Isolated myocytes: basal NOS and SOD isoform expression. A through D, Selected examples of positive labeling for indicated NOS and SOD isoforms (indirect IF) for myocytes enzymatically isolated from ferret (A1 through A6) RA, (B1 through B6) RV, (C1 through C6) LV endo, and (D1 through D6) LV epi. Calibration bar: A1=5 μm. Note that both nNOS39 and iNOS47 could be basally detected in some myocytes. E and F, Summarized mean (±SEM) results of approximate percentage myocyte expression of NOS and SOD isoforms in ferret isolated SAN, RA, RV, LV epi, and LV endo myocytes. Myocyte samples were obtained from 7 hearts for eNOS and all SOD isoforms, 5 hearts for nNOS, and 4 hearts for iNOS. Percentage expression was calculated on the following number of cell counts from each heart analyzed: SAN, 50 to 100 myocytes; RA and RV, 500 to 800 myocytes; and LV epi and LV endo, 600 to 1000 myocytes.

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-α and interferon-γ; 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 6A⇓1 through 6A4 shows EM results on the localization patterns of SOD isoforms in 60 nm thick sections obtained from ferret LV epi (Figure 6A⇓1 through 6A3) and LV endo (Figure 6A⇓4) tissue. In LV epi myocytes, MnSOD specifically localized to the mitochondria (Figure 6A⇓1), CuZnSOD was uniformly distributed throughout the myoplasm but was excluded from the mitochondria and sarcolemma (Figure 6A⇓2), and ECSOD was highly expressed and specifically localized to the sarcolemma (Figure 6A⇓3). In LV endo myocytes (Figure 6A⇓4), 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 6A⇓3 and 6A4). Similar SOD isoform expression patterns were consistently observed in LV epi and LV endo sections prepared from 3 ferret hearts.

Figure 6.
Figure 6.

A1 through A4, Subcellular localization patterns of SOD isoforms in ferret LV. Electron microscopic results on LV epi and LV endo ultrathin tissue sections either single or double labeled with indicated SOD antibodies and detected with protein A gold binding (5-, 10-, and/or 15-nm particles). M indicates mitochondria; Z, Z line; and SR, sarcoplasmic reticulum. A1, A2, and A3, LV epi sections single labeled with MnSOD antibody (10-nm particles) (A1), double labeled with MnSOD (10-nm particles) and CuZnSOD (5-nm particles) antibodies (A2), and double labeled with MnSOD (10-nm particles) and ECSOD (15-nm particles) antibodies (A3). LV endo section double labeled with MnSOD (10-nm particles) and ECSOD (15-nm particles) antibodies (A4). Please note the following: (1) the specific localization of MnSOD to mitochondria, CuZnSOD to the myoplasm, and ECSOD to the sarcolemma and (2) the much lower density of ECSOD in the sarcolemma of LV endo myocytes compared with LV epi myocytes. Magnifications: A1, ×32 000X; A2, ×43 206; and A3 and A4, ×24 000. Calibration bars: A1=40 nm, A2=60 nm, and A3 and A4=30 nm. B through D, Subcellular colocalization of eNOS and ECSOD; confocal microscopic results obtained from ferret isolated (B1 through B7) RV, (C1 through C7) LV endo, and (D1 through D7) LV epi myocytes doubly labeled (direct IF) with eNOS and ECSOD antibodies. Panels B7, C7, and D7 show the overall summed 3-dimensional view of the myocytes shown, whereas the respective rows of adjacent subpanels 1 through 6 show a series of optical Z sections (0.75 μm thickness) taken sequentially through the width of each myocyte shown in panels B7, C7, and D7 (every third Z section is shown). Note that the sequential fluorescence profiles of both eNOS (red) and ECSOD (green) (colocalization=yellow-orange) remained specifically confined to the outer regions of the myocyte image, thereby indicating colocalization to the sarcolemma. Calibration bar: B1=5 μm.

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.

Discussion

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 α 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.

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  • Heterogeneous Basal Expression of Nitric Oxide Synthase and Superoxide Dismutase Isoforms in Mammalian Heart
    Mulugu V. Brahmajothi and Donald L. Campbell
    Circulation Research. 1999;85:575-587, originally published October 1, 1999
    https://doi.org/10.1161/01.RES.85.7.575
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