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Am J Physiol Heart Circ Physiol 286: H47-H54, 2004. First published August 14, 2003; doi:10.1152/ajpheart.00730.2003
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EB2003 SYMPOSIUM
Mitochondrial Nitric Oxide

Changes in NO bioavailabilty regulate cardiac O2 consumption: control by intramitochondrial SOD2 and intracellular myoglobin

Wei Li,1 Thomas Jue,2 John Edwards,1 Xipping Wang,1 and Thomas H. Hintze1

1Department of Physiology New York Medical College, Valhalla New York 10595 and 2Department of Biological Chemistry, University of California Davis, Davis, California 95616-8635

Submitted 4 August 2003 ; accepted in final form 12 August 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this study was to investigate the significance of two intracellular scavengers of nitric oxide (NO): 1) superoxide dismutase (SOD) (SOD2) to scavenge intramitochondrial superoxide anion, and 2) cytosolic myoglobin (Mb) in the regulation of tissue O2 consumption. O2 consumption was measured in vitro using a Clark-type O2 electrode. SOD heterozygous mice (SODHZ) (n = 13) and SOD wild-type (SODWT) (n = 5) mice were used. Bradykinin (BK, 10–4 mol/l) reduced O2 consumption by 15% ± 1 in hearts of SODHZ mice, which was significantly different from SODWT (reduced by 24 ± 0.4%). Tiron significantly increased the inhibition of O2 consumption by BK in male mice from 15 ± 1% (n = 13) to 29 ± 1.2% (n = 4) at 10–4 mol/l concentration (P < 0.05). The effect of carbachol was similar to BK. S-nitroso-N-acetyl penicillamine (SNAP, 10–4 mol/l) reduced O2 consumption by 39 ± 1.3% in hearts of SODHZ mice, which was not significantly different from SODWT. But at 10–7 mol/l, SNAP caused significantly less inhibition of O2 consumption in SODHZ mice. Mb knockout (MbKO; Mb wild-type n = 6) and (MbWT) mice (n = 6) were also used. Kidney cortex was studied as the negative control because it does not contain Mb. BK (10–4 mol/l) reduced O2 consumption by 32 ± 2, 29 ± 1, and 26 ± 1% in the heart, skeletal muscle, and kidney of MbKO mice, which was also not significantly different from MbWT. SNAP (10–4 mol/l) reduced O2 consumption by 39 ± 3, 42 ± 4, and 46 ± 2% in the heart, skeletal muscle, and kidney of MbKO mice, which was also not significantly different from MbWT. NG-nitro-L-arginine methyl ester (P < 0.05) inhibited the reduction in O2 consumption induced by BK in the MbKO mouse heart (15 ± 1%), skeletal muscle (17 ± 1%), and kidney (17 ± 1%) as in the MbWT mice. These results suggest that the role of Mb as an intracellular NO scavenger is small, and the increase in mitochondrial superoxide in SODHZ mice may cause a decrease NO bioavailability and alter the control of myocardial O2 consumption by NO.

myoglobin knockout mouse; superoxide dismutase-2 heterozygous mouse; Tiron; bradykinin; S-nitroso-N-acetyl penicillamine; carbachol

CONSIDERABLE EVIDENCE suggests that reactive O2 species, such as superoxide , H2O2, and the hydroxyl radical (·OH) (38), and, more recently, peroxynitrite (ONOO–) (19), play an important role during physiological states. For instance, during normal oxidative phosphorylation, between 0.4 and 4% of all O2 consumed is converted into the superoxide free radical (15, 20, 40, 41). Free radicals have the capacity to oxidize proteins, lipids, DNA, and RNA (32, 35) as well as to chemically interact with NO. H2O2 diffuses easily out of the mitochondria, but superoxide does not because it is a charged molecule (18), and therefore it has to be scavenged within the mitochondrial matrix. Superoxide undergoes spontaneous dismutation into H2O2 and oxygen, but the dismutation reaction is greatly enhanced by manganese superoxide dismutase (SOD2, MnSOD) (13). SOD, a family of enzymes that catalyze the dismutation of to H2O2 and O2, reduces the tissue concentration of and prevents the production of ·OH and ONOO– (38). Thus, in conjunction with catalase and glutathione (GSH) peroxidase, SOD may play an important role in the host defense against O2 toxicity. Although three separate genes encode the SODs, one of them, a homotetrameric manganese form (MnSOD), is compartmentalized within the mitochondrial matrix and is coded for by the SOD2 gene (12). MnSOD plays an important role in protecting Fe-S enzymes of the citric acid cycle and electron transport chain from direct inactivation by superoxide (27, 31). SOD2 exists as a tetramer and is initially synthesized containing a leader peptide, which targets this manganese-containing enzyme exclusively to the mitochondrial spaces.

Recently, Lebowitz's laboratory (26, 30) produced MnSOD gene knockout (KO) mice by deleting exons 1 and 2 of the Sod2 gene (Sod2mlbcm). Knocking out the Sod2 gene in these mice results in a lethal cardiomyopathy. The average lifespan of SOD2 KO mice is 16 days. Western blot analysis of mitochondrial MnSOD from heterozygous animal livers revealed that MnSOD was reduced ~50% in the SODHZ animals (25). Heterozygous SOD2 (SODHZ) mice are viable and do not have any gross phenotypic abnormalities. Remmen et al. (43) demonstrated that there is no evidence that either the expression of the major antioxidant enzymes (CuZnSOD, catalase, and glutathione peroxidase) or GSH levels are upregulated to compensate for the reduced activity of MnSOD in the tissues of the SODHZ mice (43). Therefore, in SODHZ mice, the increased level of superoxide may reduce intramitochondrial NO bioavailability.

Myoglobin (Mb) is an important intracellular O2-binding hemoprotein found in the cytoplasm of vertebrate type I and IIa skeletal and cardiac muscle tissue (46), and it facilitates O2 delivery during periods of high metabolic demand. Mb contains an iron-porphyrin heme group identical to that of hemoglobin (Hb) and, like Hb, is capable of reversible oxygenation and deoxygenation. Recently, transgenic mice lacking Mb were generated. Mb knockout (MbKO) mice have a benign phenotype. They present unaltered exercise and reproductive capacity as well as cardiac and skeletal function (14).

Studies have suggested that human Mb actively participates in the regulation of NO by three distinct mechanisms, namely, oxidation, ligand binding, and through formation of biologically active S-nitroso-Mb (28). Mb is a scavenger of bioactive NO through the reaction NO with MbO2 to form metMb and nitrate (33) and may be the major mechanism of attenuating intracellular NO bioactivity in cardiac muscle. Flogel et al. (10) also suggested that Mb not only is a key element determining the magnitude of the response to NO in muscle but also plays an important role in overall NO inactivation. Thus, under physiological conditions, Mb acts as a intracellular scavenger preventing NO from reaching its intracellular receptors, perhaps in the mitochondria, in cardiomyocytes (44).

Our previous study indicated that endothelium-derived NO is an important regulator of tissue O2 consumption in cardiac and skeletal muscle. This inhibitory effect may be mediated by the formation of a nitrosyl complex with the enzyme iron-sulfur center to reduce the activity of mitochondrial enzymes, including aconitase in the Krebs cycle and complex I and complex II of the mitochondrial electron transport chain (9, 17, 21). NO primarily acts on cytochrome oxidase during which the enzyme is reversibly inhibited by NO at nanomolar concentrations by competing for the O2-binding site on the enzyme (1, 3, 4, 7). The overall goal of this study was to use these two gene knockout models, SODHZ and MbKO mice, to determine the regulation of intracellular NO bioavailability and consequently the regulation by NO of cardiac O2 consumption. We hypothesized that the regulation of cardiac O2 consumption by NO will be altered in both models by modifying the intracellular availabillity of NO.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. We interbred heterozygote SOD2 mice, which were acquired from Jackson Laboratory. Mice were genotyped by PCR of DNA. Genomic DNA was isolated from tail snips using a Wizard Genomic Purification Kit (Promega; Madison, WI). Primers unique to SOD2 and the targeting vector were as described by Jackson Laboratories. PCR was performed by using an Eppendorf thermocycler, and PCR products were visualized on a 2% agarose gel. Wild-type band of the SOD2 gene was 123 bp, and the targeting vector PCR product was 240 bp. MbKO were obtained from the University of California at Davis. All experimental mice were housed in the same type of cage and supplied with standard mouse chow and had free access to water. All protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the current National Institutes of Health and American Physiological Society guidelines for the use and care of laboratory animals.

Equipment. To measure tissue O2 consumption, a Clark-type platinum O2 electrode system was used (YSI-5331, Yellow Springs Instrument; Yellow Springs, OH). The O2 electrode was inserted into a small glass reaction chamber (3–8 ml) maintained in a constant 37°C temperature bath (YSI-5301). A built-in magnetic stirrer (480 rpm at 60 Hz) located in the bath assembly was used to keep the movement of the tissue in the incubation medium past the Teflon electrode membrane.

Preparation of cardiac muscle slices and measurement of O2 consumption. Mice were anesthetized with pentobarbital sodium (65 mg/kg), and the heart, skeletal muscle, and kidney were removed immediately. The atria, large coronary arteries, right ventricle, connective tissues, and fat were discarded. The left ventricle was bisected such that each piece of muscle contained the septum, free wall, and apex. Myocardial tissues were then incubated in Krebs bicarbonate solution containing (in mmol/l) 118 NaCl, 4.7 KCl, 1.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4, 1.1 MgSO4, and 5.6 glucose at 37°C and bubbled with 21% O2-5% CO2-74% N2 (pH 7.4) to equilibrate for at least 2 h.

O2 consumption by tissue slices was measured. At the end of the incubation period, each piece of tissue was placed in a stirred bath with 3 ml of air-saturated Krebs bicarbonate solution containing 10 mmol/l HEPES (pH 7.4). The bath was sealed using a Clark-type platinum O2 electrode. Succinate (5 x 10 –4 mol/l), a substrate for complex II, and sodium cyanide (10–3 mol/l), an inhibitor of complex IV of the electron transport chain, were added at the completion of the concentration-response curve to each agonist to confirm that the changes in O2 uptake were effects on the mitochdria. The 100% increase in O2 consumption in the presence of succinate suggests that O2 is not limiting in the bath, and the abolition of O2 consumption after the addition of sodium cyanide confirms that changes in myocardial O2 consumption were of mitochondrial origin.

Effect of bradykinin, carbachol, and S-nitroso-N-acetyl penicillamine on myocardial O2 consumption. Cumulative concentrations of the B2 kinin receptor agonist bradykinin (10–7–10–4 mol/l, Sigma) or the muscarinic agonist carbachol (10–7–10–4 mol/l, Sigma) were examined in myocardial tissues from SOD wild-type (SODWT), SODHZ, Mb wild-type (MbWT), and MbKO mice. The freely permeable superoxide scavenger tiron (10–2 M, Sigma) and the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10–4 M, Sigma) were used to investigate the role of superoxide and NO in the modulation of myocardial O2 consumption by changing the NO bioavailability. NO donor S-nitroso-N-acetylpenicillamine (SNAP) was used. We have used these techniques previously (28, 36, 48).

Transthoracic two-dimensional Doppler echocardiography studies for cardiac morphology and function and measurements of blood pressure. Transthoracic echocardiography was performed in awake trained mice using an Acuson Sequoia 256 equipped with a 15-MHz linear transducer (15L8) in a phased-array format. Generally, the heart was first imaged by the two-dimensionally guided M-mode cursor from the parasternal short-axis view. Left ventricular (LV) chamber dimension and wall thickness were measured from these M-mode tracings. End-diastolic (LVEDD) and end-systolic LV chamber dimensions (LVESD) were measured using the American Society of Echocardiography leading edge techniques (34). The measurement from three continuous cardiac cycles was averaged to establish the value for each animal. We then positioned the probe parallel to the sternum and obtained the long-axis view. From this image, we traced the LV area during the diastolic (LVDA) and end-systolic phase (LVSA) and calculated the ejection fraction (EF). Again, three consecutive cardiac cycles were used to obtain the average. The equations are the following: SF(%)= [(LVEDD–LVESD)/LVEDD] x 100 from the short axis (where SF is shortening fraction); EF(%)= [(LVDA–LVSA)/LVDA] x 100 from the long axis; LV mass = 1.055(IVST + LVEDD + PWT)3–(LVEDD)3 (mg) from the short-axis M-mode tracing (where IVST is intraventricular septum thickness and PWT is posterior wall thickness); and LV cavity volume = LVEDD3 (mm3).

Blood pressure (systolic, diastolic, and mean) was measured by an automated tail cuff (Columbus Instruments model NIBP-8) along with heart rate in the conscious state.

Calculation of tissue O2 consumption and statistical analysis. The rate of decrease in chamber PO2 was used as a measure of tissue respiration, and this was expressed as nanomoles of O2 consumed per minute per gram of tissue. All studies were performed within 30 min (about 5 min per dose of agonist), resulting in <50% of available O2 consumed. We have previously found that during this period of time, tissue O2 consumption is constant (36). The effect of drugs on tissue O2 consumption is expressed as the percent change in baseline O2 consumption.

All data are presented as means ± SE. Changes in O2 consumption induced by various agonists with and without L-NAME or in the absence or presence of Tiron were analyzed using two-way ANOVA, followed by multiple comparisons between different treatment groups using the Tukey test. Statistical significance was achieved at a probability value of <0.05. We have used all these techniques previously (8, 28, 29, 48).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cardiac phenotype in male and female SODWT and SODHZ mice. There were no significant differences between the SODWT and SODHZ mice of either gender in LV end-diastolic diameter, wall thickness, chamber cavity volume, and SF or EF (Table 1). The gross heart weights were not significantly different between SODWT and SODHZ mice of either gender (male SODWT: 143 ± 2.6 mg; male SODHZ mice: 137 ± 3.5 mg; female SODWT: 114 ± 3.0 mg; female SODHZ mice: 109 ± 4.8 mg). There was no difference in blood pressure or heart rate by the tail cuff in conscious mice (Table 1).


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  		Table 1. Hemodynamics in male and female SODWT and SODHZ mice

 

Effect of bradykinin and SNAP on tissue O2 consumption in male SODWT and SODHZ mice. Cumulative doses of bradykinin (10–7–10–4 mol/l) in SODWT mice caused dose-dependent decreases in O2 consumption (Fig. 1A). These inhibitory responses were smaller in SODHZ mice (24 ± 0.4% in SODWT vs. 15 ± 1.1% in SODHZ mice at 10–4 mol/l of bradykinin) (P < 0.05). SNAP (10–4 mol/l) decreased tissue O2 consumption by 39 ± 2.8% in SODWT mice and 39 ± 1.3% in SODHZ mice (Fig. 1B). But at 10–7 mol/l, the response to SNAP in SODHZ mice was significantly lower than SODWT mice (12 ± 1.4% vs. 5.0 ± 1.7%, P < 0.05).



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  		Fig. 1. A: effect of bradykinin on tissue O2 consumption in male SOD wild-type (SODWT) and SOD heterozygous (SODHZ) mice. Cumulative doses of bradykinin (10–7–10–4 mol/l) in SODWT mice caused dose-dependent decreases in O2 consumption. These inhibitory responses were smaller in SODHZ mice at 10–4 mol/l of bradykinin (n = 5 in wild-type mice and n = 13 in heterozygous mice). B: dose-response curve of S-nitroso-N-acetyl penicillamine (SNAP). At 10–7 mol/l, the response to SNAP in SODHZ mice was significantly lower than that in SODWT mice. Values are means ± SE. *P < 0.05, significant difference between SODHZ and SODWT in the percent change of O2 consumption.

 

Effect of bradykinin on cardiac O2 consumption in male and female SODHZ mice with and without Tiron. Bradykinin caused a concentration-dependent decrement in O2 consumption by 15 ± 1% in male SODHZ mice (Fig. 2) versus 9.3 ± 1.1% in female SODHZ mice (Fig. 2) at 10–4 mol/l concentration (P < 0.05) and by 12 ± 1.1% in SODHZ mice vs. 5.3 ± 1.6% in female SODHZ mice at 10–5 mol/l concentration (P < 0.05). Tiron significantly increased the inhibition of O2 consumption in male mice from 15 ± 1% (n = 13) to 29 ± 1.2% (n = 4) at 10–4 mol/l concentration (P < 0.05) and from 12 ± 1.1% to 21 ± 2% at 10–5 mol/l concentration (P < 0.05). In female mice, Tiron also increased the inhibition of O2 consumption from 9.3 ± 1.1% (n = 5) to 25 ± 1.6% (n = 5) at 10–4 mol/l concentration (P < 0.05) and from 5.3 ± 1.6% to 12 ± 2% at 10–5 mol/l concentration (P < 0.05, Fig. 2).



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  		Fig. 2. Bradykinin reduces cardiac O2 consumption in male and female SODHZ mice with and without Tiron. Bradykinin caused a concentration-dependent decrement in O2 consumption male SODHZ mice (A) and in female SODHZ mice (B) at 10–4 mol/l concentration (P < 0.05). Tiron significantly increased the inhibition of O2 consumption in male mice and female mice at the two highest doses of bradyninin. Values are means ± SE. *P < 0.05, significant difference with Tiron in the percent change O2 consumption.

 

Effect of carbachol on cardiac O2 consumption in female SODHZ mice. Carbachol caused concentration-dependent decreases in O2 consumption in tissues from female SODHZ mice. Tiron significantly increased the inhibition of O2 consumption from 10 ± 1.4% (n = 5) to 21 ± 1.2% (n = 5) at 10–4 mol/l concentration (P < 0.05) and from 6.7 ± 1.2% to 13 ± 1.6% at 10–5 mol/l concentration (P < 0.05) (Fig. 3).



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  		Fig. 3. Graph showing dose-response curve of carbachol on cardiac O2 consumption in female SODHZ mice. Carbachol caused concentration-dependent decreases in O2 consumption in tissues from female SODHZ mice. Tiron significantly increased the inhibition of O2 consumption at 10–4 mol/l concentration (P < 0.05) and at 10–5 mol/l concentration (P < 0.05). Values are means ± SE. *P < 0.05, significant difference with Tiron in the percent change in O2 consumption.

 

SOD2 protein level in the liver of SODHZ and SODWT mice. The quantitation by Western blot of SOD2 protein levels in the livers of SODHZ and SODWT mice are shown in Fig. 4. There is an ~60% decrease in the SOD2 protein level in SODHZ mice compared with the SODWT mice.



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  		Fig. 4. Graphs showing the SOD2 protein level in livers of SODHZ and SODWT mice. There is ~60% decrease in the SOD2 protein level in SODHZ mice compared with the SODWT mice. Values are means ± SE. *P < 0.05.

 

Effect of bradykinin and SNAP on O2 consumption in MbWT and MbKO mice in heart, skeletal muscle, and kidney cortex. Bradykinin caused a dose-dependent decrease of O2 consumption in the cardiac muscle, skeletal muscle, and kidney cortex to a similar degree in both MbWT and MbKO mice (Fig. 5). Bradykinin-induced reduction in O2 consumption was attenuated by L-NAME (10–4 mol/l, control: 27 ± 0.9% vs. L-NAME: 15 ± 1.1% in heart tissue; control: 27 ± 2.7% vs. L-NAME: 17 ± 1.8% in skeletal muscle; 26 ± 1.2% vs. L-NAME: –17 ± 0.9% in kidney cortex; P < 0.05). The responses to SNAP were not different between MbWT and MbKO mice in the heart muscle, skeletal muscle, and kidney cortex. These responses were not affected by L-NAME (Fig. 6).



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  		Fig. 5. Graphs showing the effect of bradykinin on cardiac O2 consumption in wild-type myoglobin (Mb) (MbWT) and Mb knockout (MbKO) mice in heart muscle (A), kidney cortex (B), and skeletal muscle (C) in the presence and absence of NG-nitro-L-arginine methyl ester (L-NAME). Bradykinin caused dose-dependent decreases of O2 consumption in cardiac muscle, kidney cortex, and skeletal muscle to a similar degree in both MbWT and MbKO mice. Bradykinin-induced reduction in O2 consumption was attenuated by L-NAME. Values are means ± SE. *P < 0.05, significant difference with L-NAME in the percent change in O2 consumption.

 


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  		Fig. 6. Graphs showing the effect of SNAP on cardiac O2 consumption in MbWT and MbKO mice in heart muscle (A), kidney cortex (B), and skeletal muscle (C) in the presence and absence of L-NAME. There were no differences. Values are means ± SE. *P < 0.05, significant difference with L-NAME in the percent change in O2 consumption.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrate that the NO inhibition of O2 consumption was attenuated in SODHZ mice, and Tiron restored this response. Bradykinin caused less inhibition of O2 consumption in female SODHZ mice than that in male SODHZ mice. The cardiac phenotype is normal in SODHZ mice of either gender. There was no significant difference in the regulation of O2 consumption by NO between control and MBKO mice.

In the current study, bradykinin, cabarchol, and SNAP, an exogenous NO donor, inhibited tissue O2 consumption, which is consistent with our previous findings (28, 36, 48). In the mouse, we have already demonstrated that bradykinin and cabarcol caused endogenous NO release through B2 kinin receptor-mediated and muscarinic receptor-mediated mechanisms, respectively, because HOE-140 and atropine blocked the inhibitory effect of bradykinin and cabarchol (48). In B2 kinin receptor and eNOS knockout mice (28, 29), bradykinin had no effect on O2 consumption (8).

We noted that bradykinin caused less inhibition of tissue O2 consumption in SODHZ than in SODWT mice. In SODHZ mice, there is 30–80% reduction of SOD2 protein in the liver resulting in reduced superoxide metabolism and increased binding of superoxide with NO. Studies have shown that superoxide scavenges NO to form ONOO–, a shorter-lived and less potent vasorelaxant than NO. Therefore, the increase in superoxide production may cause the decrease in NO bioavailability (23). This change in NO bioavailability could account for the small effect in SODHZ mice. Very interestingly, at low doses, the response to SNAP is significantly smaller in SODHZ mice compared with the SODWT mice, which may suggest that the level of superoxide was high enough to scavenge exogenous NO, if NO is present at low concentrations but not sufficient to surmount high levels of NO.

To further test whether superoxide is indeed involved, we used the superoxide scanvenger Tiron. As expected, Tiron significantly enhanced the bradykinin-induced inhibition of O2 consumption in both male and female SODHZ mice, and Tiron also enhanced the reduction of tissue O2 consumption by carbachol in female SODHZ mice. The change with Tiron strongly suggests that superoxide is binding NO.

When we compared the response to bradykinin in male versus female SODHZ mice, we found that at high doses, bradykinin caused significantly less inhibition of O2 consumption in female versus male mice. This suggests that the bioavailability of NO in female mice is lower than that in male mice, and the oxidative stress may be higher. The mechanism underlying this difference is unknown.

A recent study (33) has indicated that Mb reacts with NO to form metMB in the perfused myocardium and can play a role in scavenging NO. This has been confirmed by several studies (5, 10). We used MbKO mice to investigate the role of intracellular Mb in mediating the actions of NO on the mitochondria. Very unexpectedly, in both control and MbKO mice, bradykinin reduced tissue (heart muscle and skeletal muscle) O2 consumption to a similar degree, and the NOS inhibitor L-NAME attenuated this response to a comparable degree. Kidney cortex containing no Mb was used as negative control, and the response was not different. SNAP reduced tissue O2 consumption in the heart muscle, skeletal muscle, and kidney cortex in both MbKO and control mice. Accordingly, we did not find evidence that Mb can scavenge NO in contrast to previous studies (5, 10). Flogel et al. (10) showed that for Mb to scavenge NO, the NO level needs to be higher than 1 µm. Under our experimental conditions, the level of NO was most likely in the range of hundreds of nanomolar. This may explain the discrepancies. A recent study by this group (47) also showed that induction of inducible NO synthase-generated NO could be inactivated by Mb, but again these are most likely high levels of NO.

Homozygous mutant mice (SOD knockout) with no MnSOD activity on a CD-1 background have been shown to develop extensive myocardial damage and die of dilated cardiomyopathy shortly after birth even in room air (27). SODHZ mice with a 50% reduction in myocardial MnSOD activity did not develop any myocardial injury in a 100% O2 environment (39). In our study, the cardiac structure and function as assessed by echocardiography were not different in the SODHZ mice. This is consistent with another study, which showed that SODHZ mice developed and survived normally in room air with no demonstrable histological or ultrastructural abnormalities in the lung or heart (39). Considerable evidence suggests that reactive O2 species, such as , H2O2, ·OH (38) and, more recently, ONOO (19), play an important role in hyperoxia-induced tissue injury. The endogenous production of reactive O2 species due to normal physiological processes is a major limiter of lifespan (2, 6). During normal oxidative phosphorylation, between 0.4 and 4% of all O2 consumed is converted into the superoxide free radical (15, 20, 40, 41). O2 toxicity is mediated through reactive O2 species, and hyperoxia increases the mitochondrial production of superoxide and H2O2 (11). H2O2 diffuses easily out of mitochondria, but superoxide does not because it is a charged molecule (18), and therefore it has to be scavenged within the mitochondrial matrix. Superoxide undergoes spontaneous dismutation into H2O2 and superoxide, but the dismutation reaction is greatly enhanced by manganese superoxide dismutase (SOD2, MnSOD) (13).

The antioxidant defense system is a highly complex and integrated system that functions to protect cells against the potentially harmful effects of reactive O2 species produced as a result of aerobic metabolism. This system is composed of both antioxidant enzymes and nonenzymatic low-molecular-weight antioxidant molecules such as GSH. SOD, a family of enzymes that catalyze the dismutation of to H2O2 and O2, reduces the tissue concentration of and prevents the production of ·OH and ONOO (38). Thus, in conjunction with catalase and GSH peroxidase, SOD may play an important role in the host defense against O2 toxicity. The SODs are the first and most important line of antioxidant enzyme defense systems against reactive oxygen species and particularly superoxide anion radicals, by enzymatically scavenging superoxide anions converting them to H2O2. Three separate genes encode the SODs: a homotetrameric CuZn-containing form, which is located in the extracellular space; a cytosolic homodimeric CuZn form (Cu-Zn/SOD) coded for by the SOD1 gene; and a homotetrameric manganese form (MnSOD), which is compartmentalized within the mitochondrial matrix and is coded for by the SOD2 gene (12). MnSOD plays an important role in protecting Fe-S enzymes of the citric acid cycle and electron transport chain from direct inactivation by superoxide (27, 31). SOD2 exists as a tetramer and is initially synthesized containing a leader peptide, which targets this manganese-containing enzyme exclusively to the mitochondrial spaces. SOD2 has been shown to play a major role in promoting cellular differentiation and tumorgenesis (37) and in protecting against hyperoxia-induced pulmonary toxicity (45). In contrast to other major antioxidant enzymes, e.g., Cu-Zn/SOD, catalase, and GSH peroxidase, the expression of MnSOD is modulated by a variety of physiological and environmental factors, suggesting that MnSOD may play a role in physiological processes other than the antioxidant defense system (43).

Recently, two laboratories have independently produced MnSOD gene knockout mice: Li and associates (27) deleted exon 3 of the Sod2 gene Sod2mlucsf, whereas Lebowitz and co-workers (26) deleted exons 1 and 2 of the Sod2 gene Sod2mlbcm. The deleted exon 3 in the first model codes for the amino acids responsible for dimer-tetramer formation and for manganese binding at the active site of the enzyme. The importance of SOD2 function in the mammalian organism was confirmed by disruption of the SOD2 gene, which turns out to be lethal for mice due to neurodegeneration, damage to the heart (26), and also liver complications, metabolic acidosis, and early neonatal death (27). The mice we used in this study were originally made by Lebowitz and co-workers (26). Although knocking out the SOD2 gene is lethal, SODHZ mice are viable and do not have any gross phenotypic abnormalities. Adult SODHZ mice with half the normal MnSOD activity are not more susceptible to 100% O2 than wild-type mice (24, 39). Studies showed that in mice, only 50% of the MnSOD activity may be sufficient for normal resistance to 100% O2 toxicity (39). There is no evidence that either the expression of the major antioxidant enzymes (Cu-Zn/SOD, catalase, and GSH peroxidase) or GSH levels are upregulated to compensate for the reduced activity of MnSOD in the tissues of the SODHZ mice (43). GSH levels are reduced in several tissues of the SODHZ mice, which is consistent with greater oxidative stress in these tissues (42). They did have demonstrable biochemical abnormalities (42). On an inbred C57BL/6 background, there was a 30% reduction in complex I and aconitase activities in liver mitochondria and a 44% decrease in state 3 respiration with duroquinol as the substrate (22).

Superoxide scavenges NO to form ONOO–, a shorter-lived and less potent vasorelaxant than NO; the increase in superoxide production may cause the decrease in NO bioavailability that has been observed in the aorta of stroke-prone spontaneous hypertensive rats (23). Therefore, in SODHZ mice, the increased level of superoxide may decrease NO bioavailability.

Mb is an important intracellular O2-binding hemoprotein (46), and it facilitates O2 delivery during periods of high metabolic demand. Mb contains an iron-porphyrin heme group and is a short-term O2 reservoir in exercising skeletal muscle and in the beating heart. Recently, transgenic mice lacking Mb were generated. MbKO mice are normal and have unaltered exercise and reproductive capacity as well as cardiac and skeletal function (14). MbKO mice manifest adaptations in the skeletal muscle that include a fiber-type transition (type I to type II in the soleus muscle), increased expression of the hypoxia-inducible transcription factors hypoxia-inducible factor (HIF)-1{alpha} and HIF-2 (endothelial PAS domain protein), stress proteins such as heat shock protein-27, and the angiogenic growth factor vascular endothelial growth factor (soleus muscle) as well as increased NO metabolism (extensor digitorum longus). Those resulting changes in angiogenesis, NO metabolism, and vasomotor regulation are likely to account for preserved exercise capacity of these mice (16).

Several studies have shown that Hb is an NO scavenger (28). NO binds readily to the thiol and metal groups of the Hb. These authors demonstrated that native Hb induced a greater constrictor response and inhibited NO-mediated relaxation. Studies showed that human Mb actively participates in the regulation of NO by three distinct mechanisms, namely, oxidation, ligand binding, and through formation of biologically active S-nitroso-Mb (28). Mb reacts with NO because MbO2 combines with NO to form metMb and nitrate in cardiac muscle. Flogel et al. (10) also suggested that Mb not only is a key element determining the magnitude of the response to NO in muscle but also plays an important role in overall NO inactivation. Thus under physiological conditions, Mb acts as a intracellular scavenger preventing NO from reaching its intracellular receptors in cardiomyocytes (44).

Our previous study indicated that endothelium-derived NO is an important regulator of tissue O2 consumption in cardiac and skeletal muscle. This inhibitory effect may be mediated by the formation of a nitrosyl complex with enzyme iron-sulfur center to reduce the activity of mitochondrial enzymes, including aconitase in the Krebs cycle and complex I and complex II of the mitochondrial electron transport chain (9, 17, 21). NO primarily acts on cytochrome oxidase, during which the enzyme is reversibly inhibited by NO at nanomolar concentrations by competing with the O2-binding site on the enzyme (1, 3, 4, 7).

The overall goal of this study was to use these two gene knockout models, SODHZ and MbKO mice, to determine the importance of these mechanisms in the regulation of intracellular NO bioavailability and consequently the regulation by NO of cardiac O2 consumption. We hypothesized that NO bioavailabity would decrease in the SODHZ and actually be greater in the MbKO mouse due to altered intracellular availabillity of NO.

In conclusion, in SODHZ mice, NO bioavailability is decreased by the scavenging effect of superoxide on NO. This is particularly evident in studies using Tiron, which enhanced the bioavailability of NO in the SODHZ mouse heart. Our data suggest that Mb did not scavenge NO at the low (endogeneous) concentrations generated by bradykinin or carbachol and not even at potentially high concentrations generated during the decomposition of SNAP. Our study suggests that inside the cell there are several mechanisms that control NO bioavailabililty to regulated mitochondrial function and tissue O2 consumption.


   ACKNOWLEDGMENTS
 
We thank K. Rafalski for genotyping the SOD mice.

GRANTS

W. Li was supported by a predoctoral fellowship from the New York State Affiliate of the American Heart Association (215297T). This work was part of W. Li's Doctoral Thesis in the Department of Physiology New York Medical College. This work was supported by National Heart, Lung, and Blood Institute Grants PO-43023, HL-50142, HL-61290 (to T. H. Hintze), and GM-58688 (to T. Jue).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. H. Hintze, Dept. of Physiology, New York Medical College, Valhalla, NY 10595 (E-mail: Thomas_Hintze{at}NYMC.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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